Dissertations / Theses on the topic 'Microfluidics paper-based analytical device'

The addition of powered components for active assays into paper-based analytical devices opens new opportunities for medical and environmental analysis in resource-limited applications. Current battery designs within such devices have yet to adopt a ubiquitous circuitry material, necessitating investigation into printed circuitry for scalable platforms. In this study, a microfluidic battery was mated with silver-nanoparticle conductive ink to prototype an all-printed sensing platform. A multi-layer, two-cell device was fabricated, generating 200 μA of direct electrical current at 2.5 V sustained for 16 minutes with a power loss of less than 0.1% through the printed circuitry. Printed circuitry traces exhibited resistivity of 75 to 211 10-5 Ω m. Resistance of the printed traces increased upwards of 200% depending on fold angle and directionality. X-ray diffraction confirmed the presence of face-centered cubic silver after sintering printed traces for 30 minutes at 150°C in air. A conductivity threshold was mapped and an ink concentration of 0.636 μL mm-3 was identified as the lower limit for optimal electrical performance.

Microfluidic paper based analytical devices (microPADs) are a novel platform for point of care (POC) diagnostics. Limitations of reagent shelf life have been overcome with the introduction of reagent pencils as a method for solid-based reagent deposition. While useful, little work has been reported on the characterization and optimization of reagent pencils. Herein, an investigation on reagent pencil composition and efficiency is conducted via colorimetric release profile tests utilizing an erioglaucine disodium salt that yields a quantifiable blue colored product in the presence of water. Within this work, an investigation on the molecular weight dependence, polymer chain end functionality, and polymer-graphite ratio was conducted to determine the most desirable parameters in reagent pencil composition. Further, the effects of enzyme stability in the presence of poly(ethylene glycol) (PEG) is investigated. To show the versatility of reagent pencils, a novel reagent pencil incorporating a stimuli responsive polymer, poly(N-isporopylacrylamide) (PNIPAM) was developed. In this work, PNIPAM’s lower critical solution temperature (LCST) was manipulated with various salt solutions to control fluid flow both laterally and vertically through various microPAD designs. It was found that, while PNIPAM successfully blocked or retarded fluid flow in microPADs, the effect was limited when DI H2O wash solutions were run prior to salt solutions. To counteract this, PNIPAM was successfully covalently bound to alkene modified chromatography paper via thiolene click chemistry to reinforce solution wash tolerance.

Monitoring blood coagulation while a patient is on cardiopulmonary bypass (CPB) is critical in preventing clots from arising in the bypass machine and consequently being sent into the patient’s bloodstream. Current methods used to monitor blood coagulation such as Activated Clotting Time (ACT) yield results that do not correlate coagulation time to heparin or protamine dosage and will typically take at least 400 s to yield a result that is safe to initiate bypass. Microfluidic paper-based analytical devices (μPAD) are advanced sensors based on a wide range of recently developed techniques for complex analytical methods. In this research, a point-of-care (POC) sensor was developed based on techniques adapted from lateral flow and µPAD. The effects of varied dosages of heparin and protamine were observed using this POC µPAD and an accompanying Raspberry pi-based monitoring device. Paper microfluidic channels were printed on nitrocellulose paper with a wax pattern. Human whole blood was added to an absorbent fiber glass sample pad preloaded with known amount of heparin or protamine. By having this absorbent pad on the inlet of the channel, the blood sample is able to travel through the channel via capillary flow. Significantly different (p < 0.05) rates of flow between blood samples with different doses of heparin and protamine show that the device can monitor the extent of coagulation and patient-specific responses to each drug. Thus a low-cost device was built that monitors the extent of blood coagulation and allows for individualized dosing of heparin and protamine in as little at 20 s and no more than 180 s.

N,N′-methylenebisacrylamide-crosslinked poly(N-isopropylacrylamide), also known as P(NIPAM), was developed as a fluid delivery system for use with microfluidic paper-based analytical devices (microPADs). MicroPADs are postage-stamp-sized devices made out of paper that can be used as platforms for low-cost, simple-to-use point-of-care diagnostic assays. P(NIPAM) is a thermally responsive polymer that absorbs aqueous solutions at room temperature and will expel the solutions to microPADs when heated. The fluid delivery characteristics of P(NIPAM) were assessed, and P(NIPAM) was able to deliver multiple solutions to microPADs in specific sequences or simultaneously in a laminar-flow configuration. P(NIPAM) was then shown to be suitable for delivering four classes of reagents to microPADs: small molecules, enzymes, antibodies and DNA. P(NIPAM) successfully delivered a series of standard concentrations of glucose (0 – 5 mM) to microPADs equipped to perform a colorimetric glucose assay. The results of these tests were used to produce an external calibration curve, which in turn was used to determine accurately the concentrations of glucose in sample solutions. P(NIPAM) successfully delivered fluorescein-labeled IgG and fluorescein-labeled oligonucleotides (20 base pairs) to microPADs in a variety of concentrations. P(NIPAM) also successfully delivered horseradish peroxidase (HRP) to microPADs, and it was determined that HRP could be stored in P(NIPAM) for 35 days with minimal loss in activity. The combination of P(NIPAM) with microPADs will allow for more complex assays to be performed with minimal user input, will facilitate the preparation of external calibration curves in the field, and may be useful in extending the shelf life of microPADs by stabilizing reagents.

Paper Based Microfluidic Devices (microPADs) are a new platform for point-of-care diagnostic assays for use in resource-limited settings. These devices rely typically on enzymatic assays to produce their results, which makes them susceptible to degradation when exposed to extreme environmental conditions such as high temperature. In order to overcome this limitation, this research project focused on investigating the use of polymers instead of enzymes to detect analytes on microPADs. Polymer-bound boronic acid, a glucose and pH sensitive polymer, was incorporated into microPADs in order to develop a chronometric, paper-based glucose assay. The polymer was tested with both lateral and vertical flow microPADs made from three different types of paper, and several different methods of incorporating the polymer into the devices were also explored. While some devices appeared to show a trend in signal versus concentration of glucose, none of the results were statistically significant due to the large standard deviations in the signal. Upon further analysis of the results, the overall conclusion was that the devices were not sensitive enough to detect glucose in the range of concentrations that would be practical for clinical diagnostic applications.

The recent growing interest and development of microfluidic paper-based analytical devices (μPADs) for point-of-care (POC) testing in human health in low-resource settings has great potential for the exploitation of these technologies in animal disease diagnosis. Sheep scab is a highly infectious, widespread and notifiable disease of sheep, which poses major economic and welfare concerns for the UK farming industry. The possibility of diagnosing sheep scab at the POC is, consequently, very important to controlling this disease. The overall aim of this project was, therefore, to develop μPADs based on a novel method of fabrication, in order to translate the existing lab-based sheep scab ELISA (Pso o 2) and a biomarker test for haptoglobin (Hp) into paper-based ELISA (P-ELISA), to enable POC diagnosis of this animal disease. In Chapter 3, the novel fabrication method is described, in Chapters 4 and 5, the translation of the lab-based ELISAs (Hp and Pso o 2 respectively) are explained and in Chapter 6 the development of a μPAD for incorporation of the POC tests into a multiplexed, rapid assay is covered. Experiments showed that both ELISAs were successfully transferred onto paper and that the devices developed were suitable for POC testing. This study has resulted in a novel fabrication method for μPADs, in successfully translated existing ELISAs to P-ELISA and in novel solutions for the POC diagnosis of an important veterinary disease.

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care diagnostic tests for use by untrained users with potential applications in healthcare, environmental monitoring, and food safety. These devices can be developed for a multitude of different tests, many of which employ enzymes as catalysts. Without specialized treatment, some enzymes tend to lose their activity when stored on microPADs within 48 hours, which is a major hurdle for taking these types of devices out of the laboratory and into the real world. This work focused on the development of simple methods for stabilizing enzymes by applying polymers to chromatography paper. The longterm stabilization was exlored and SU-8 of various concentrations was found to stabilize horseradish peroxidase for times in excess of two weeks. A variety of microPAD fabrications, enzyme dispensing methods, and substrate delivery techniques were explored.

The activity carried out during my PhD was principally addressed to the development of portable microfluidic analytical devices based on biospecific molecular recognition reactions and CL detection. In particular, the development of biosensors required the study of different materials and procedures for their construction, with particular attention to the development of suitable immobilization procedures, fluidic systems and the selection of the suitable detectors. Different methods were exploited, such as gene probe hybridization assay or immunoassay, based on different platform (functionalized glass slide or nitrocellulose membrane) trying to improve the simplicity of the assay procedure. Different CL detectors were also employed and compared with each other in the search for the best compromise between portability and sensitivity. The work was therefore aimed at miniaturization and simplification of analytical devices and the study involved all aspects of the system, from the analytical methodology to the type of detector, in order to combine high sensitivity with easiness-of-use and rapidity. The latest development involving the use of smartphone as chemiluminescent detector paves the way for a new generation of analytical devices in the clinical diagnostic field thanks to the ideal combination of sensibility a simplicity of the CL with the day-by-day increase in the performance of the new generation smartphone camera. Moreover, the connectivity and data processing offered by smartphones can be exploited to perform analysis directly at home with simple procedures. The system could eventually be used to monitor patient health and directly notify the physician of the analysis results allowing a decrease in costs and an increase in the healthcare availability and accessibility.

The activity carried out during my PhD was principally addressed to the development of portable microfluidic analytical devices based on biospecific molecular recognition reactions and CL detection. In particular, the development of biosensors required the study of different materials and procedures for their construction, with particular attention to the development of suitable immobilization procedures, fluidic systems and the selection of the suitable detectors. Different methods were exploited, such as gene probe hybridization assay or immunoassay, based on different platform (functionalized glass slide or nitrocellulose membrane) trying to improve the simplicity of the assay procedure. Different CL detectors were also employed and compared with each other in the search for the best compromise between portability and sensitivity. The work was therefore aimed at miniaturization and simplification of analytical devices and the study involved all aspects of the system, from the analytical methodology to the type of detector, in order to combine high sensitivity with easiness-of-use and rapidity. The latest development involving the use of smartphone as chemiluminescent detector paves the way for a new generation of analytical devices in the clinical diagnostic field thanks to the ideal combination of sensibility a simplicity of the CL with the day-by-day increase in the performance of the new generation smartphone camera. Moreover, the connectivity and data processing offered by smartphones can be exploited to perform analysis directly at home with simple procedures. The system could eventually be used to monitor patient health and directly notify the physician of the analysis results allowing a decrease in costs and an increase in the healthcare availability and accessibility.

The aim to provide preventive healthcare and high quality medical diagnostics and treatment to an increasingly ageing population caused a rapidly increasing demand for point-of-care diagnostic devices. Disposables have an advantage over re-usable units as cross-contamination is avoided, no cleaning and sterilising of equipment is required and devices can be used out of centralised laboratories. To remain cost-effective, costs for disposables should be kept low. This makes polymer materials an obvious choice. One method for the realisation of fluidic micro devices is the stacking of several layers of microstructured polymer films. Reel-to-reel manufacturing is a promising technique for high-volume manufacturing of disposable polymer bio-analytical devices. Polyethylene terephthalate (PET) and cycloolefin copolymer (COC) were selected as suitable polymer substrate materials and polydimethyl siloxane (PDMS) as membrane layer. Bonding of polymer films with the help of adhesives carries the risk of channel blocking. Despite this drawback, no other method of bonding PDMS to a structural layer could be identified. Bonding with solvents avoids channel blocking issues, but adversely affects biocompatibility. Thermal diffusion processes enable bonding of COC and PET without the use of any auxiliary material. The extensive process times requires for thermal diffusion bonding can be considerably shortened by pre-treating the material with plasma or UV exposure. Welding with the use of a laser energy absorbing dye was demonstrated to be particularly suitable for selective bonding around channels and reservoirs. None of the assessed bonding methods provide a generic solution to all bonding applications. Instead, the selection of an appropriate technique depends on the intended application and the required level of biocompatibility. Since this selection has implications on the feasibility and reliability of microfluidic structures on the device, design rules which ensure design for production have to be established and followed.

Paper (and other cellulose-based materials such as cotton thread and fabrics) are underexploited as materials for the construction of “high-tech” and “lab-on-a-chip” devices. One major drawback of paper is its tendency to absorb water from the environment and, with wetting, to change its mechanical properties; other challenges relate to control over the attachment of molecules (e.g. antibodies, DNA) and cells on its surface, and to the addition of electronic function. The goal of this thesis is to develop paper as a substrate for a range of applications— microfluidics, substrates for electronic systems and MEMS, low-cost diagnostics, cell biology, and optics. The approach involves chemically modifying the surface of the paper to provide new functions without altering any of its defining properties: mechanical flexibility, foldability, light weight, gas permeability, and low cost. The first part of my thesis describes the modification of paper by silanization with organosilanes such as alkyl- and fluoroalkyl trichlorosilanes in the gas phase. Here, silanization is used to lower the surface free energy of the paper and to minimize the tendency of paper to absorb liquids and vapors, and especially water. Chapter 1 and Appendix 3 demonstrate that the combination of long fluoroalkyl chains of grafted siloxanes with the micro-scale roughness and porosity of paper yielded a material that is omniphobic (both hydrophobic and oleophobic), while preserving the properties of mechanical flexibility and low resistance to transport of gas of the untreated paper. Appendix 3 shows that features of omniphobic paper can be used to construct microtiter plates and liquid-filled gas sensors using standard paper folding techniques, while Appendix 4 shows that new type of microfluidic device fabricated by carving microchannels into the surface of omniphobic paper. The resulting devices have open, unobstructed channels (with dimensions as small as 45 μm) and thus exhibit fluid dynamics similar to conventional PDMS-based microfluidics, but are much lighter and have the potential to be much less expensive than PDMS-based devices. The second part of my thesis is focused on engineering the surface of paper to enable efficient immobilization of capture and target molecules for bioanalysis. In one approach, described in Appendix 5, we exploit the ease with which the surface chemistry of paper (i.e. the surface of the cellulose fibers making up the paper) can be modified, in order to enhance the immobilization of antibodies and antigens on the surface of the paper via hydrophobic interactions, while preventing the wicking of the fluids into the paper substrate. As an application in low-cost diagnostics, we describe a low-cost electrochemical device for ELISA intended for use in resource-limited settings. In a second approach, described in Chapter 2, we developed of an efficient procedure for assembling microarrays of ssDNA and proteins on paper, at the lowest practical cost. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper, and proceeds to generate ssDNA arrays that, through hybridization with complementary strands of DNA, are capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The third part of my thesis describes the simple, inexpensive fabrication of electrodes for paper-based electrochemical systems. A first method describes, in Appendix 6, the development of inkjet printing as a method for high resolution printing of conductive patterns on omniphobic “RF” paper, both to extend its promise as a substrate for paper electronics, and to enable us to integrate it into our program in low-cost, paper based diagnostics. A second method, described in Chapter 3, circumvents the need for printing, and instead focuses on the fabrication and reconfiguration of simple, versatile, and inexpensive electroanalytical devices in which conventional stainless-steel pins—in unmodified form or after coating with a carbon paste—are used as electrodes.
Chemistry and Chemical Biology

The purpose of this thesis was to demonstrate device functionality of 3-D paper-based, multiplex platforms, µPADs, without the use of coupling agents between layers. Previously, these platforms were fabricated with double-sided tape and cellulose powder to try to augment proper fluid routing, but difficulties with this method occurred. An acrylic housing unit with strategically placed pressure tabs was designed to aid horizontal and vertical fluid routing through the platform, thus eliminating the inconsistencies associated with coupling agents. Channel characterization studies, a COMSOLTM simulation, and development time studies were performed to aid device design and demonstrate device functionality. The implementation of this µPAD platform as a diagnostic instrument was validated via lateral flow immunoassays utilizing both biotinylated antibodies and biotinylated aptamers as capture reagents. Successful detection of the target analyte, IgE, as well as successful fluid routing through multiple layers of membrane was demonstrated by immunoassays performed on 3-D, multiplex platforms. Another important result determined the aptamers’ ability to detect IgE to be statistically the same as the antibodies’ ability; thus confirming aptamers as viable capture reagent alternatives to antibodies in lateral flow assays. Overall, this research project was performed to develop and validate via experiment a prototype paper-based microfluidic diagnostic device, µPAD, with the capability to detect multiple biomarkers on one platform.

Les S-nitrosothiols (RSNOs) sont considérés comme des stocks circulant de monoxyde d'azote (NO) et qui ont de nombreux rôles in vivo. Une variation de la proportion des taux de RSNOs a été démontrée dans de nombreuses maladies. Il est donc important de pouvoir identifier et quantifier chaque RSNO dans les fluides biologiques pour la réalisation de diagnostics médicaux. Il devient alors intéressant de développer des outils analytiques pour la détermination des RSNOs, en utilisant de faibles volumes d'échantillons biologiques. Ce travail de thèse a ainsi été orienté vers le développement d'outils analytiques miniaturisés pour l'analyse des RSNOs dans les fluides biologiques, en se focalisant sur la conception de micro-dispositifs (laboratoires sur puce), intégrant toutes les étapes de l'analyse, à savoir l'injection, la séparation, la décomposition et la détection sur un seul et même dispositif pour l'identification et la quantification des RSNOs. Pour cela, chaque étape a dû être optimisée. Ainsi, une meilleure compréhension de la réactivité des RSNOs, en terme de voies de décomposition et de cinétique, a été étudiée en développant deux méthodologies basées sur l'électrophorèse capillaire (CE) couplée soit à la spectrométrie de masse (MS) soit à une détection par mesure de conductivité sans contact à couplage capacitif (C4D). Par la suite, les conditions de décomposition et la détection sensible du NO libéré ont été réalisées en utilisant des microcapteurs électrochimiques à NO. Sur la base des résultats obtenus, deux stratégies originales ont été développées pour la détection de la totalité des RSNOs présents dans le plasma (i) via la décomposition des RSNOs en utilisant des nanoparticules d’or couplées à des microcapteurs NO et (ii) via la conception d’un dispositif miniaturisé de diagnostic sur papier. Finalement, grâce à l’optimisation des étapes de décomposition, de séparation et de détection, une étude préliminaire a été menée pour concevoir une micropuce d’électrophorèse intégrant la décomposition des RSNOs et une détection électrochimique afin de quantifier indépendamment différents RSNOs
S- nitrosothiols (RSNOs) are considered as biological circulating stock of nitric oxide (NO) that have many roles in vivo. The variation of RSNOs proportion has been recognized in many diseases, so that the identification and quantitation of each RSNO in biological fluids is of prime importance. There is thus interest for the development of analytical tools for their determination, using low biological sample volumes. This PhD work was thus orientated towards the development of miniaturized analytical tools for the analysis of RSNOs in biological fluids, with a focus on microdevices (lab-on-a-chip), by integrating the injection, separation, decomposition and detection steps for the simultaneous identification and quantitation of various RSNOs. To this aim, a better understanding of RSNO reactivity, in terms of decomposition, was necessary and was assessed by developing two methodologies based on capillary electrophoresis (CE) coupled to different detection techniques: mass spectrometry (MS) and capacitively coupled contactless conductivity detection (C4D). Then, the conditions for RSNOs decomposition and further sensitive detection of released NO by miniaturized electrochemical NO-sensors were determined. Finally, two original strategies were developed for the detection of the total amount of RSNOs in plasma (i) decomposition using gold nanoparticles and (ii) conception of miniaturized paper-based point of care device. Thanks to the optimization of decomposition, separation and detection steps, preliminary work was conducted to develop a microchip electrophoresis coupled to RSNOs decomposition to quantify separately the different RSNOs

The purpose of this thesis project is to demonstrate and evaluate an enzyme-linked immunosorbent assay (ELISA) on a paper microfluidic device platform. The integration of ELISA technology onto paper microfluidic chips allows for a quantitative detection of stroke biomarkers, such as glial fibrillary acidic protein (GFAP). Dye experiments were performed to confirm fluid connectivity throughout the 3D chips. Several chip and housing designs were fabricated to determine an optimal design for the microfluidic device. Once this design was finalized, development time testing was performed. The results confirmed that the paper microfluidic device could successfully route fluid throughout its channels at a reasonable rate. For the biochemistry portion of this thesis project, antibodies were selected to target the intended stroke biomarker: GFAP. However, due to antibody pairing complications, the protein chosen for this project was natural human cardiac troponin T, which is elevated in the bloodstream of patients who have suffered a stroke. Several antibody experiments were performed to help finalize the procedure for performing an ELISA on the paper chip. The final antibody experiment was able to demonstrate that a paper microfluidic device utilizing ELISA techniques can successfully detect a stroke biomarker at physiologically relevant concentrations. Overall, this project supported the ability to accurately and effectively diagnose stroke in a timely manner through the use of a paper microfluidic device.

Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (𝜇PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored.
Ph. D.

碩士
國立高雄海洋科技大學
水產食品科學研究所
106
Nitrite is one of the commonly, use preservatives in the food system. Nitrite in the meat products red color and inhibit the growth of Clostridium botulinum. Therefore, is necessary to use nitrite in the food products to ensure safety and maintain color. However, nitrite is a known carcinogen and possesses a potential risk. The traditional analyses of nitrite, include chromatography, electrochemical approach, spectral analysis and capillary electrophoresis etc. Using microfluidic paper-based analytical devices（μPADs）has many advantages such as simple, non-expensive, and fast. Therefore, the purpose of this study is to use microfluidic technique to develop a quick nitrite assay system based on the chromomeric reaction on the paper. In this study, the paper chip coated with a coloring agent combining microfluidic technique. Color density and red-green-blue（RGB）values were measured by an application program（APP）in cellular phone and regular cellular phones could be conveniently used for this assay. The standardization was conducted by different concentrations of nitrite. Results showed tightly negative correlation between the nitrite concentrations and color density （R2=0.99） and the detection ranges were 10-40 and 50-90 ppm. In addition, the paper chip maintained its effectiveness for more than one month when stored at 4˚C. Nitrite was artificially added into meat products, vegetables, edible swallow's nests, and fish farm water and checked by this assay system. In the recovery testing, recovery rate of meat products, vegetables, edible swallow's nests, and fish farm water were 96.86%-108.41%, 101.29%, 101.27%, and 96.11% respectively. Results showed the range of recovery rates were in the standard of development of food chemical assay method （80%-115%）.The samples were delivered to the Food Safety Center of Uni-President Company and the results were cross-compared. Results of cross-comparison showed that the deviation of μPADs system was lower. Thus, this μPADs system has the advantages of simple, fast, and cheaper than traditional methods. In addition, this system was also stable and sensitive. Thus, it possesses a great commercial value.

Nonostante il notevole ampliamento della rete di controlli, finalizzati a ridurre la diffusione a livello mondiale di composti illeciti di droga, l'abuso di sostanze psicotrope continua ad essere un fenomeno in aumento. I sequestri di cocaina, eroina, morfina e cannabis hanno mostrato una tendenza costante dal 2003 al 2012. I sequestri di stimolanti amfetamino-simili hanno seguito la stessa tendenza, fino al 2010, mentre 2010-2012 i sequestri sono stati triplicati. Inoltre, alcuni laboratori basano la propria attività sullo sviluppo di nuove sostanze psicoattive (NPSS) che non rientrano nella convenzione internazionale di controllo della droga. Secondo l'Ufficio delle Nazioni Unite contro la Droga e il Crimine (UNODC), le NPSs sono definite come "sostanze d'abuso ... che non sono controllate dalla Convenzione sugli stupefacenti del 1961 o la Convenzione del 1971 sulle sostanze psicotrope .... In questo contesto, il termine 'nuovo' non si riferisce necessariamente a nuove invenzioni, ma alle sostanze che sono recentemente disponibili ". La diffusione dei composti psicotropi non comuni è già una questione di tener conto. Il numero delle NPSs sul mercato globale è più che raddoppiato nel periodo 2009-2013. Negli ultimi anni la rilevazione di droghe illecite é stata diretta verso l'impiego di metodi analitici rapidi e precisi. La drammatica crescita della tecnologia permette di eseguire misurazioni analitiche impiegano volume dell'ordine dei nanolitri e picolitri. Questo progetto di ricerca mira a dimostrare l'affidabilità dei sistemi microfluidici, come strumenti di analisi a fini tossicologici forensi. Diversi dispositivi microfluidici possono essere utilizzati per perseguire scopi differenti; nel presente progetto saranno esposti tre diverse strategie finalizzate a rilevare nuove sostanze psicoattive. Qui, il termine nuovo, è utilizzato per indicare l'uso voluttuario di un farmaco comunemente somministrati per scopi anestetici, la ketamina, e di un composto antitosse che viene utilizzato in molti farmaci da banco, il destrometorfano. La prima strategia è stata focalizzata sulla sviluppo di un dispositivo microfluidico basato su carta mediante rivelazione colorimetrica per l'analisi di ketamina e altre droghe di abuso. Una varietà di metodi presuntivi sono stati sviluppati, compresi test tubes, microscopia, TLC, GC e IR, ma molti di questi test richiedono tecnici specializzati, o come nel caso dei test tubes, non sono in grado di determinare contemporaneamente una grande varietà di composti in un unico step. Per quanto a nostra conoscenza questo lavoro presenta un unico processo per l'esecuzione di più test contemporaneamente. Il test può rilevare un'ampia varietà di analiti utilizzando pochi microgrammi di campione. Analisi semiquantitativa è possibile anche utilizzando uno smartphone e software semplice. Il metodo proposto non richiede persone altamente qualificate o strumentazione costosa, e può essere eseguita in loco rendendo possibile una risposta analitica durante le azioni di polizia, servizi di frontiera, e sicurezza negli aeroporti. La seconda parte del progetto ha sottolineato lo sviluppo e la validazione di un semplice metodo di separazione chirale per identificare e quantificare gli enantiomeri del metorfano, e loro principali metaboliti, utilizzando elettroforesi capillare mediante selettori chiarali costituiti da ciclodestrine (CD-CZE). Per quanto a nostra conoscenza, i metodi disponibili sono basati su complessi metodi MEKC e ciclodestrine (CD-MEKC) o metodi che richiedono colonne cromatografiche costosi. Nel 2013, Koo C et al. sviluppato un GC - MS utilizzando colonna achirale, ma il metodo non è stato applicato a campioni di sangue. Il metodo proposto ha consentito di risolvere destrometorfano da levometorfano in sequestri di eroina e di eseguire l'identificazione chirale e la quantificazione di enantiomeri del metorfano e loro principali metaboliti , destrorfano e levorfanolo, nel sangue prelevato in sede autoptica, con una sensibilità accettabile. La sezione finale del progetto è stato centrato su un studio preliminare finalizzato allo sviluppo di un metodo per rilevare la ketamina, basato su elettroforesi capillare e polimeri a stampo molecolare (MIP-CZE). I risultati preliminari sono stati ottenuti utilizzando la tecnica HPLC-MS per caratterizzare la capacità di interazione in termini di recupero estrazione di ketamina e comuni droghe di abuso. La complessitá del processo ci ha indirizzato a sviluppare un metodo rapido alternativo in grado di valutare la capacità di interazione dei polimeri a stampo molecolare da utilizzare per analisi CZE. I risultati preliminari hanno dimostrato l'adeguatezza e l'affidabilità del elettroforesi capillare per caratterizzare le proprietà dei MIP in formato nanoparticellare (NP MIP). Questo studio pone le basi per la possibilità di utilizzare NP MIP come selettori ad alta specificità da utilizzare in analisi CZE.
Notwithstanding the remarkable amplification of the network of controls, finalized to reduce the worldwide spread of illicit drug compounds, the abuse of psychotropic substances continues to be a rising phenomenon. The seizures of cocaine, heroin and illicit morphine and cannabis showed a constant trend from 2003 to 2012. The seizures of amphetamine-type stimulant followed the same trend until to 2010, while from 2010 to 2012 the seizures were trebled. In addition, some laboratories base their activity on developing of New Psychoactive Substances (NPSs) that fall outside international drug control convention. According to the United Nations Office on Drugs and Crime (UNODC), NPSs are defined as “substances of abuse…that are not controlled by the 1961 Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances…. In this context, the term ‘new’ does not necessarily refer to new inventions but to substances that have recently become available”. The spreading of uncommon psychotropic compounds is already a matter to take account. The number of NPSs on the global market more than doubled over the period 2009-2013 1. In recent years the detection of illicit drugs has been directed toward a rapid and accurate recognition analytical methods. The dramatic growth of the technology allows performing of analytical measurements employing volume in the range of the nanoliter and picoliter. This research project seeks to demonstrate the reliability of microfluidic systems, as analytical tools for forensic toxicological purposes. Several microfluidic devices can be used to pursue different aims; in the present project will be exposed three different strategies finalized to detect new psychoactive substances. Here, the term new, is utilized to indicate the voluptuary use of a medicine commonly administered for anesthetic aims, the ketamine, and the reveler utilization of an antitussive compound which is used in many over-the-counter drug, the dextromethorphan. The first strategy was focused on the developing of a set of paper microfluidic colorimetric tests for the analysis of ketamine and other common drugs of abuse. A variety of presumptive methods have been developed, including spot tests, chemical microscopy, TLC, GC and IR but many of these tests require skilled handlers, or like spot tests are incapable of simultaneously determining a wide variety of compounds in a single tube analysis step. To the best of our knowledge this work presents a unique process for running multiple assays simultaneously. The test can detect a wide variety of analytes using few micrograms of sample. Semiquantitative analysis is also possible using a smartphone and simple software. The proposed method doesn’t require highly qualified persons or expensive instrumentation, and it can be performed on-site enabling a prompt analytical response during police actions, border services, and airport security. The second part of the project emphasized the development and validation of a simple chiral separation method to identify and quantify the enantiomers of methorphan, and their main metabolites, by using cyclodextrin-assisted capillary zone electrophoresis (CD-CZE). To the best of our knowledge, the available methods are based on complex method cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) 2 or methods which require expensive chromatographic columns 3. In 2013, Koo C et al. developed a GC/MS by using achiral column, but the method was not applied to blood samples 4. The proposed method allowed to resolve dextromethorphan from levomethorphan in seizures of heroin and to perform the chiral identification and the quantification of methorphan enantiomers and their main metabolites, dextrorphan and levorphanol, in post-mortem blood, with an acceptable sensitivity. The final section of the project was centered on preliminary study finalized to the development of a method to detect ketamine, based on molecularly imprinted polymer-assisted capillary zone electrophoresis (MIP-CZE). The preliminary results were obtained by using the technology HPLC-MS to characterize the capacity of interaction in terms of extraction recovery of ketamine and common drugs of abuse non-structure related to ketamine. The tediousness of the process addressed us to develop an alternative rapid method able to evaluate the capacity of interaction of the molecularly imprinted polymers to use in CZE analysis. Preliminary results demonstrated the suitability and the reliability of the capillary electrophoresis to characterize the binding properties of molecularly imprinted polymers nanoparticle format (NP MIP). This study lays the groundwork for the possibility to utilize NP MIP as high-specificity selector to use in CZE analysis.

abstract: DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system.
Dissertation/Thesis
Presentation for Lin Gan's thesis defense (orginally in pptx exported in PDF)
Doctoral Dissertation Chemistry 2015

De todos os catiões presentes no corpo, o magnésio é o segundo mais importante catião e o quarto mais prevalente. Doenças que envolvem o magnésio são classificadas em dois grupos: hipomagnesemia (défice de magnésio) e hipermagnesemia. Desta forma, a determinação de magnésio despertou grande interesse, porque auxilia no contexto clínico e em pesquisas epidemiológicas. Portanto, o objetivo deste trabalho foi desenvolver um dispositivo microfluídico analítico em papel (μPAD) para quantificar magnésio em amostras salivares. Neste caso em concreto, o μPAD baseia-se numa reação colorimétrica entre o magnésio e o eriocrómio de cianina, formando uma cor laranja/avermelhada intensa. Após a reação, é necessário utilizar um scanner de mesa para obter uma imagem de alta resolução da zona de deteção do μPAD para determinar a intensidade de cor laranja/avermelhada de cada unidade de teste, medida através do software Image J. Sob condições ótimas, o método para o μPAD proposto foi caracterizado por um intervalo de calibração para a concentração de magnésio entre 0.082 – 0.247 mmol/L. Os limites de deteção e quantificação foram 0.062 mM e 0.081 mM, respetivamente. O gasto dos reagentes, eriocrómio cianina, NH4Cl e NH4OH foram 0.043 mg, 1.62 mg e 13.0 mg por curva de calibração. O gasto da solução padrão/amostra foi 120 μl por cada determinação.
Of all the cations in the body, magnesium is the second most important intracellular cation and the fourth most prevalent. Disorders involving magnesium are categorized into two groups: hypomagnesemia (magnesium deficiency) and hypermagnesemia. In this way, the determination of magnesium has aroused great interest, because it helps in the clinical context and epidemiological research. Therefore, the objective of this work was to develop a microfluidic paper-based analytical device (μPAD) for the quantification of magnesium in saliva samples. In this case, the μPAD is based on the colorimetric reaction between magnesium and eriochrome cyanine to form an intense orange/reddish dye. After the reaction, it is necessary to use a flatbed scanner to obtain a high-resolution image of the detection zone for determination the intensity of the orange/reddish colour within each detection zone measured with Image J software. Under the optimum conditions, the proposed μPAD method was characterized by a linear calibration range for magnesium concentration 0.082 – 0.247 mmol/L. The detection and quantification limits were 0.062 mM and 0.081 mM, respectively. The reagents, eriochrome cyanine, NH4Cl and NH4OH consumption were 0.043 mg, 1.62 mg and 13.0 mg per calibration curve and the sample consumption was 120 μl per eachdetermination.

碩士
東吳大學
化學系
107
The first part is "Development of a portable fluorescent detector", with the increasing number of aging population, our research target has become to develop a simple and a small equipment to achieve real-time monitoring health condition. This article uses 3D printing technology to create a portable fluorometer that can be combined with mobile phones. The portable fluorometer includes a first body part, a second body part and a support body part. The design with the advantages of small size, easy storage, less interference from the scattering light, and low-cast. An approved patent application has been provided by the Intellectual Property Office, and the patent certificate number is: M566327. The second part is "Design Satereogram", The main content is to draw, Three-dimensional figures by using the 3D software. A large amount of text information in books, newspapers and magazines often combines pictures to assist in explanations, increasing people's understanding of words and enhancing memory. Besides, with the advancement of technology, the development of computer graphics software has advanced from the basic Microsoft Paint, Pinta, Artweaver, etc. to 3D Builder and Blocks CAD which can draw 3D objects. Therefore, we put 3D pictures into the paper to increase the reader's understanding of the experiment and device. We take " Determination of Nitrite Ion in environment analysis with a Paper-Based Microfluidic Device" and "Detecting Glucose Levels in Blood Plasma and Artificial Tear by Au(I) Complex on the Carbopol Polymer: A Microfluidic Paper-Based Method". The third part is " Development of a paper-based near-infrared emitting Iridium(III) complex for glucose detection in human blood ". Near-infrared (NIR) dyes have attracted increasing attention for their potential applications in optical imaging in vivo and medical diagnosis. The diagnostic window falling in the NIR range from 650 to 900 nm surpasses the visible region and allows for bioimaging with minimal interference from tissue autofluorescence, reduced light scattering, and high tissue penetration. Ir(III) complexes with mixed ligands in particular have been the subject of considerable interest because of their bright photoluminescence efficiency, long luminescence lifetime, and excellent color tuning. The strong spin–orbit coupling induced by iridium(III) promotes an efficient intersystem crossing from the singlet to the triplet excited state manifold, which then facilitates strong electroluminescence by harnessing both singlet and triplet excitons after the initial charge recombination. Despite intense research using transition metal complexes for glucose detection, application of a glucose biosensor utilizing NIR-emitting Iridium complexes is few. For pursuing high-efficiency NIR-emitting iridium(III) complexes as glucose sensing, we cooperate with Prof. Sergey P. Tunik in Russia. NIR-emitting iridium(III) complex, Ir2, which exhibits NIR emission with a maximum peak at 620 nm. In this study, optical microfluidic paper analytical devices (μ-PADs) for glucose detection from whole blood samples with a small sample volume have been developed on a single paper. In the proposed method, a mushroom-shaped analytical device contains a sample inlet zone and a detection zone. When blood is dripped onto the inlet region of a μPAD, the plasma diffuses to the detection region. The detection region is implanted with a metallic three-dimensional (3D) metal complex hydrogel vehicle. The gel vehicle consists of a complex that responds to oxygen changes and glucose oxidase (GOx) immobilized inside the gel as a bioactivity preservative. The phosphorescence of the complex is enhanced by oxygen consumed by detection of glucose. The influences of alginic acid concentration, calcium chloride concentration, pH and acid buffer concentration are also discussed.

Nos últimos anos, os ensaios de dispositivos analíticos baseados em papel apresentaram um crescimento significativo na indústria de testes de diagnóstico point-of-care (POC) sendo considerados uma tecnologia sólida e robusta. Entre as principais vantagens, estes dispositivos caracterizam-se pela simplicidade, precisão relativa, tempo de retorno rápido, fácil utilização e preço acessível. Tendo em conta estas vantagens, estes testes suscitaram o interesse não só da academia, mas também da indústria levando ao aparecimento de uma grande gama de aplicações (e.g. testes de gravidez, testes de glucose, marcadores de enfarte e cancro, doenças infecciosas ...), posicionando o mercado de testes baseados em dispositivos analíticos de papel num valor de vários milhões de dólares. Esta dissertação pretende explorar uma nova abordagem para a combinação de testes baseados em papel com um espectrómetro NIR portátil, com particular foco em: i) projetar plataformas de teste mais compactas e simples de manusear; e ii) obter resultados de classificação e quantificação em soluções aquosas de alta e baixa viscosidade. De forma a conseguir obter uma plataforma com as características identificadas acima, foi necessária a combinação de vários componentes de forma orquestrada numa única plataforma. Os conceitos teóricos que permitiram suportar o desenvolvimento deste sistema e que justificam o método utilizado baseiam-se em: i) apresentar um estudo abrangente de dispositivos analíticos baseados em papel; ii) fornecer conceitos teóricos relativos à espetroscopia NIR e quimiometria; iii) apresentar o desenvolvimento do dispositivo de teste com integração na forma de realização final; e, finalmente, iv) exibir os resultados projetados no fluxo de trabalho. Resultados experimentais utilizando o suporte final combinado com uma plataforma protótipo que foi concebida para servir como prova de conceito na aplicação em soluções de baixa viscosidade mostraram resultados promissores na classificação e quantificação de soluções diluídas de glucose e vinho. A aplicação de um PAD ligeiramente diferente, alterado na sua camada alfa, apresentou resultados surpreendentes em amostras de sangue preservadas com EDTA para análise de hemograma. O sistema integrado foi capaz de classificar diferentes amostras de sangue e também quantificar parâmetros cruciais como hemoglobina e eritrócitos, quando comparado com a tecnologia de ponta utilizada em análises do mesmo tipo.
In recent years, the application of microfluidic paper-based analytical devices (PADs) has seen a tremendous growth on the diagnostic industry. It is considered to be a highly promising class of point-of-care (POC) diagnostic tests and represents a multibillion-dolar market on its own. This development was motivated by the combination of unique properties of paper-based materials, which includes its simplicity, relative accuracy, fast results, low price and user friendliness. This technology has been used not only in the academic context but also to support a wide range of commercial applications including glucose tests, pregnancy tests, monitoring and testing of cardiac panels in emergency conditions, infectious disease screening, cancer marker screening and drugs abuse testing. This thesis aims at exploring a new approach for combining paper-based analytical devices with a NIR portable spectrometer, with particular focus on: i) designing compact test platforms that are easier to use and handle; and ii) achieve classification and quantification results for aqueous solutions, with low and high viscosity properties. In order to achieve the platform with the properties indicated above, it was necessary to combine several components in a single orchestrated architecture. To pave the way for the development of this project, this work provides: i) a comprehensive study of paper analytical devices; ii) a background theory of NIR spectroscopy and chemometrics; iii) presents the development of the multilayer test strip with an integration in the final embodiment; and, finally, iv) describes the results of the engineered workflow. Experimental results using the final embodiment combined with a prototype platform that was designed to serve as proof-of-concept for the applicability of the envisioned setup in low viscosity solutions have shown promising results in classification and quantification of glucose and wine diluted solutions. The application of a slightly different PAD, altered in the alpha layer, have shown surprising results on blood samples, preserved with EDTA for a blood count analysis. The integrated system was able to classify different blood samples and also quantify crucial parameters as hemoglobin and erythrocytes, when compared to the gold standard technology.

碩士
國立臺灣大學
化學工程學研究所
102
In this work, we first demonstrated an all-dry, top-down, and one-step rapid process to fabricate paper-based microfluidic devices using fluorocarbon plasma polymerization. This process is able to create fluorocarbon-coated hydrophobic patterns on filter paper substrates while maintaining the trench and detection regions intact and free of contamination after the fabrication process, as confirmed by ATR-FTIR and XPS. We have shown that the processing time is one critical factor that influences the device performance. For the device fabricated with a sufficiently long processing time (180 s), the sample fluid flow can be well confined in the patterned trenches. By testing the device with 800 μm channel width, a sample solution amount as small as 4.5 μL is sufficient to perform the test. NO2&;#8722; assay is also performed and shows that such a device is capable for biochemical analysis. In the second part of this master thesis, a portable microplasma generation device (MGD) operated in ambient air is introduced for making a microfluidic paper-based analytical device (μPAD) that serves as a primary healthcare platform. By utilizing a printed circuit board fabrication process, a flexible and lightweight MGD can be fabricated within 30 min with ultra low-cost. This MGD can be driven by a portable power supply (less than two pounds), which can be powered using 12V-batteries or AC-DC converters. This MGD is used to perform maskless patterning of hydrophilic patterns with sub-mm spatial resolution on hydrophobic paper substrates with good pattern transfer fidelity. Using this MGD to fabricate μPADs is demonstrated. With a proper design of the MGD electrode geometry, μPADs with 500 μm-wide flow channels can be fabricated within 1 min and with a cost of less than $USD 0.05/device. We then test the μPADs by performing quantitative colorimetric assay tests and establish calibration curve for detection of glucose and nitrite. The results show a linear response to glucose assay for 1-50 mM and nitrite assay for 0.1-5 mM. The low cost, miniaturized, and portable MGD can be used to fabricate μPADs on demand, which is suitable for in-field diagnostic tests. We believe this concept brings impact to the field of biomedical analysis, environmental monitoring, and food safety survey.

The work presented in this thesis describes research towards design, and novel means of fabrication of distance-based microfluidic paper-based analytical devices (μPADs), and development of selected applications for (bio)chemical point-of-care analysis. Chapter 1 provides insights into the principles and evolution of μPADs, particularly those based on the distance-based detection method. Chapter 2 introduces a new facile fabrication technique for (distance-based) μPADs using a low-cost desktop electronic craft plotter/cutter. This simple approach enabled rapid prototyping of μPADs by coupling a wax printer with a plotter/cutter, enabling the desired cutting and reagent deposition steps being performed in a fully integrated and geometrically aligned manner along with the wax-printing step. Chapter 3 extends the development of this fabrication method and further investigates the deposition characteristics and performance of this novel approach, demonstrating its capability for deposition of in-house formulated reagents upon μPADs with various designs, while the deposition volume could be precisely quantified gravimetrically. Chapter 4 demonstrates the effect of the μPAD device geometry on the distance signal obtained from the fabricated distance-based μPADs. It is shown that implementation of asymmetrical geometry microfluidic paper channels (varying width instead of the typical rectangular channels) improved analytical parameters significantly. This is demonstrated with the instrumentation-free determination of chloride via trapezoidal distance-based μPADs from as little as 5 μL sample volume, a key requirement for analysis of chloride in sweat as a basis of diagnosis of cystic fibrosis. In chapter 5, the stable immobilization of chromogenic reagents upon paper, being a significant issue in regard to distance-based detection, is addressed. Herein, anion exchange filter paper is introduced as a new substrate for fabrication of μPADs, providing immobilization of water-soluble anionic reagents upon the paper surface and consequently allowing the development of distance-based assays without being compromised by the sample fluid washing away effect, otherwise limiting the choice of reagents to only water-insoluble ones. This extends the applicability of the distance-based detection method to assays which involve water-soluble anionic chromogenic reagents, not readily immobilized on the standard filter paper. Finally, Chapter 6 of this thesis discusses the limitations of (distance-based) μPADs and provides further insight and direction for future research.

碩士
國立成功大學
化學工程學系
104
In this study, we have proposed and demonstrated a relatively simple and fast technique, i.e. filtration-assisted lithography (FilL) to fabricate paper-based microfluidic devices. The filter paper was first patterned with the tape, followed by dispensing polymer solution on top of the paper and turning on vacuum pump. It was found that the polymer was retained inside the filter paper at the designated locations to form the barrier, i.e. the channel wall. The minimum channel width is approximately 1000 m. When using 22 wt% polymethylmethacrylate solution with 120,000 molecular weight, the paper-based microfluidic devices can be fabricated in approximately 1 min.

碩士
國立中興大學
生醫工程研究所
103
A novel oral fluid testing platform based on microfluidic paper‐based analytical devices (µPADs) is proposed for on-site high-throughput monitoring of driving under the influence of drug (DUID). A key advantage of the proposed technology is the ability to simultaneously and effectively measure low concentration of ketamine in oral fluid without the need of complex equipment, power sources and long processing time. The detection system will provide key elements including (a) adequate, consistent and high recovery oral fluid collection without dilution, (b) selection of antibodies for high-specific immunoassay screening of specific drug classes, (c) high sensitive and high throughput µPAD fabrication and characterization for capturing and concentrating abused drugs in oral fluid and (d) surface modification for high density of different types of antibodies binding on cellulose substrates, offering a robust and rapid solution for on-site drug screening. As a result, a ketamine assay with a detection limit of 10 ng/mL, and a single assay can be completed in 6 min.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006.
Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3766. Adviser: Ralph G. Nuzzo. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

碩士
國立中興大學
生醫工程研究所
101
Colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for inorganic mercury detection in environment. Unmodified gold nanoparticles, 3-mercaptopropionic acid (MPA) and detection single-stranded deoxyribonucleic acid (ssDNA) are used to achieve rapid mercury ion sensing without complicated and time-consuming thiolated or other surface-modified probe preparation processes. To eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the turnaround time is only1 hour, and the detection limit adopting MPA-Au NPs and ssDNA-Au NPs mixtures are 750 nM and 100 nM, respectively. We believe the proposed platform possesses the potential for on-site mercury pollution monitoring in resource constrained settings.

Nos últimos anos, investigadores, com o auxílio de novas tecnologias, têm trabalhado para o desenvolvimento de técnicas e dispositivos de diagnóstico e tratamento mais práticos e económicos. Neste trabalho foram desenvolvidos dois dispositivos microfluídicos analíticos baseados em papel (μPADs) para a determinação dos aniões nitrito e nitrato em amostras de saliva humana, para auxílio no diagnóstico de doenças associadas à sua presença. Para desenvolver estes μPADs foram realizados vários estudos de desenho e construção, incluindo um teste de interferências e estudos de estabilidade. A estrutura final do μPAD para a determinação de anião nitrito consistiu em duas camadas de discos de papel de filtro com 9.5 mm de diâmetro numa bolsa de plastificação, em que a última camada continha 5 μL de reagente de Griess. Este μPAD permitiu a determinação de nitrito num intervalo de 5 – 220 μM, cujos limites de deteção e quantificação foram 0.05 μM e 0.17 μM, respetivamente. A estrutura final do μPAD para a determinação de anião nitrato consistiu em três camadas de papel de filtro numa bolsa de plastificação, em que a primeira camada continha zinco em pó e a última camada 10 μL de reagente de Griess. Este μPAD permitiu a determinação de anião nitrato num intervalo de 0.2 – 1.2 mM, cujos limites de deteção e quantificação foram 0.08 mM e 0.27 mM, respetivamente. Ambos os μPADs se mostraram estáveis quando armazenados em vácuo (durante pelo menos 7 dias no caso do μPAD de nitrito, e durante no máximo 3 dias no caso do μPAD de nitrato) e, após colocação da amostra, os μPADs de nitrito e nitrato poderiam ser digitalizados até 4 e 2 horas depois, respetivamente. Por último, para validar este método, os resultados obtidos com o μPAD de nitrito foram comparados com os correspondentes aos obtidos pelo método colorimétrico de referência, e não foram encontradas diferenças estatisticamente significativas entre os dois métodos. Logo, foi possível concluir que os μPADs desenvolvidos demonstraram possuir propriedades promissoras para a determinação de NOX em amostras de saliva, principalmente porque são dispositivos sensíveis, portáteis, simples e económicos, que custam menos de 50 cêntimos cada.
In the last few years, researchers, with the help of new and advanced technologies, have been working towards the development of more practical and more affordable, diagnostic and treatment devices and techniques. In this work, two different Microfluidic Paper-based Analytical Devices (μPADs) were developed for the determination of nitrite and nitrate in human saliva samples to aid in the diagnosis of some diseases and health conditions associated with these ions. To develop these nitrite and nitrate μPADs, several studies were performed to optimize the design and construction, including an interference assessment and stability studies. The final structure of developed μPAD for the nitrite determination consisted of two layers of 9.5 mm diameter filter paper disks within a plastic laminating pouch, in which the bottom layer contained 5 μL of Griess reagent. This μPAD allowed a nitrite determination in a range of 5 - 220 μM with limits of detection and quantification of 0.05 μM and 0.17 μM, respectively. The nitrate μPAD final structure consisted of three layers of filter paper, also within a plastic laminating pouch, in which the top layer contained the zinc powder and the bottom layer contained 10 μL of Griess reagent. This μPAD allowed a nitrate determination in the range 0.2 – 1.2 mM with limits of detection and quantification of 0.08 mM and 0.27 mM, respectively. Both of the μPADs were stable when stored in vacuum (the nitrite μPAD for at least 7 days and the nitrate μPAD for a maximum of 3 days) and, after the sample placement, the nitrite and nitrate μPADs could be scanned within the first 4 and 2 hours, respectively. Finally, to validate this method, nitrite μPAD measurements were compared with the ones obtained from the standard colorimetric method and there were no statistically significant differences between these two methods. So, it was possible to conclude that the developed μPADs exhibited promising properties for NOX determinations in saliva samples, especially because they are sensitive, portable, simple and affordable devices that cost less than 50 cents each.

abstract: Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type is altered upon environmental stimulations, influencing the bacterial survival and virulence within the host tissue. Therefore, it is important to explore methods to identify the sub-populations of Salmonella. This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges. Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2015

Thread-based microfluidic analytical devices (μTADs) have attracted growing interest in medical, biological, and environmental analysis, due to their low-cost, biocompatibility, ease of assembly, and low-reagent consumption applications. The hydrophilic properties, mechanical strength, and soft and flexible nature of threads enable the fabrication of μTADs without hydrophobic wax barriers as required in paper-based microfluidics and are compatible with modification or functionalisation under wet conditions. Additionally, preparation of μTADs with simple configurations is possible by using threads with different surface chemistries via cutting, knotting, or sewing. Ambient ionization mass spectrometry (AIMS) provides direct ionization of analytes on the surface and subsurface of samples under ambient conditions, with minimal sample manipulation. Over the past few years, a series of ambient ionization techniques have been developed to enhance the capability of AIMS and expand its applications. These techniques have been widely applied in forensics, drug development, and medical diagnostics. However, the lack of simple and rapid sample clean-up and separation procedures typically limits the application of AIMS for the analysis of trace levels of analytes in complex samples like biological fluids, containing high levels of interfering species. This thesis has focused on the combination of μTADs, for the pretreatment of complex biological samples, and desorption electrospray ionization mass spectrometry (DESI-MS), for the rapid analysis of complex biological samples. The combination of μTADs with AIMS can address the limitations of singly applying AIMS to the analysis of complex samples. Additionally, μTADs-AIMS has significant future potential in developing on-site analytical devices by utilising miniature mass spectrometers. Chapter 1 reviews the recent advances and applications of μTAD in analytical sciences. It gives an overall introduction of the fabrication techniques and detection methods of μTAD systems, followed by the classification of the applications. The pros and cons of μTADs and future trends in this area are also discussed. Chapter 2 discusses the advances of AIMS with specific focus on the new generation of ionization techniques (like DESI, direct analysis in real time, DART, extractive electrospray ionization, EESI, paper spray ionization, PSI, etc.), developed to enhance the capabilities of AIMS and to expand its applications. A major focus of this Chapter is on the use and role of nanomaterials in AIMS techniques to further improve their performance and application. In Chapter 3, the research aimed to explore and develop a simple and inexpensive threadbased sample preparation and concentration method and its coupling with DESI-MS for rapid ‘on-thread’ electrofluidic analysis. However, the detection of water-containing samples is a challenge for DESI-MS. Therefore, herein a platform for thread-based isoelectric focusing (TB-IEF) was 3D-printed, optimised, and applied for the separation and focusing of three model proteins from aqueous media. After the TB-IEF separation and concentration process, the thread with focused analytes was dried through an applied voltage and directly subjected to the DESI-MS source. This combination delivered a novel and low-cost approach for the sample pretreatment and focusing of amphoteric solutes in complex aqueous samples, with direct ‘on-thread’ ambient MS detection. This setup was applied for the separation and focusing of bovine serum albumin, R-phycoerythrin, myoglobin (MB), and cytochrome c. Successful separation and focusing was achieved within 30 min. A 10-fold increase in the DESI-MS response was achieved following the TB-IEF preconcentration, whilst simultaneously isolating the target solutes from their sample matrix. The TB-IEF-DESI-MS technique is mainly for the analysis of proteins and peptides, therefore, the development of a similar method for small molecules is an area of interest. Hence, in Chapter 4, the focus of the research was to integrate a thread-based isotachophoresis (TBITP) sample treatment approach with DESI-MS for the analysis of small target molecules in complex biological samples. However, to couple the TB-ITP technique with DESI-MS, it was necessary to focus the analytes at a predetermined position, without band diffusion and postfocussing. To achieve this, an on-thread trap was proposed, a simple trapping knot (from a different thread material) tied to the separation thread at a predetermined position. The proposed TB-ITP-DESI-MS setup and methodology was applied for the clean-up, preconcentration, and determination of alkaloids (coptisine, berberine and palmatine) in biological fluids. This system enabled the focusing and rapid analysis of the analytes of interest in complex matrices that were otherwise challenging for direct ambient MS. A single string of Nylon 6 thread was used as the electrophoresis substrate and a cotton knot, tied to the nylon thread, was used as the trapping zone of the ITP focused model analytes. Compared to the direct DESI-MS detection, the signal-to-noise ratio (S/N) for coptisine, berberine and palmatine obtained using the proposed method was increased 11.6-, 5.5- and 5.7-fold, respectively, due to the reduced matrix interference and focusing. Although the TB-ITP was successfully coupled with DESI-MS with the aid of a trapping knot, the applicability of this method was limited by the trapping capability of available thread materials. Accordingly, a sorptive nanomaterial was used to functionalize the trapping knot to improve the efficiency and selectivity. For this purpose, in Chapter 5, a nanomaterial-assisted TB-ITP-DESI-MS setup was developed for the clean-up, focusing, and trapping of target compounds in biological samples. Nylon thread was coated with graphene oxide (GO) by using bovine serum albumin (BSA) as the linker. The GO coated thread was then tied on the electrophoresis substrate (nylon thread) to trap the TB-ITP focused solutes in a predetermined position, followed by exposing the trapped solutes (on the dried knot) to the DESI source. The performance of this setup and strategy was verified by the analysis of model alkaloids in urine samples. Compared with the traditional direct DESI-MS analysis, the sensitivity of this method for coptisine, berberine and palmatine was enhanced 7.8-, 9.2-, 9.0-fold, respectively. Compared to the method developed in Chapter 4, this method delivered higher signal intensities and better selectivity. Most importantly, this research has opened up a new perspective to use a wide range of nanomaterials with different features, to achieve higher selectivity for the TBITP separation and trapping of targeted molecules. Finally, Chapter 6 summarises the results and findings of this project and discusses the future trends in thread-based electrofluidic systems. This project investigated different strategies for sample pretreatment using electrofluidic thread-based systems followed by DESIMS detection for rapid and sensitive detection. The performance and applicability of the proposed systems were demonstrated by analysing biological samples.

In this dissertation, three different microfluidic devices with bioanalytical applications are presented. From chapter to chapter, the bioanalytical focus will gradually become the development of a point-of-care sensor platform able to yield a reliable and quantitative response in the presence of the desired target. The first device consists of photolithographically-patterned gold on glass bipolar electrodes and PDMS Y-shaped microchannels for the controlled enrichment, separation from a mixture, and delivery of two charged dyes into separate receiving microchannels. The principle for the permanent separation of these dyes is based on the concept of bipolar electrochemistry and depended on the balancing/unbalancing of convective and electromigrating forces caused by the application of a potential bias, as well as the activation/deactivation of the bipolar electrodes. Two different bipolar electrode configurations are described and fluorescence is used to optimize their efficiency, speed, and cleanliness of delivery. The second device is a DNA sensor fabricated on paper by wax printing and folding to form 3D channels. DNA is detected by strand-displacement induced fluorescence of a single-stranded DNA. A multiplexed version of this sensor is also shown where the experiment results in “OR” and “AND” Boolean logic gate operations. In addition, the nonspecific adsorption of the reagents to cellulose is studied, demonstrating that significant reduction of nonspecific adsorption and increased sensitivity can be achieved by pre-treating the substrate with bovine serum albumin and by preparing all analyte solutions with spectator DNA. The third device, also made of paper, has a novel design and uses a versatile electrochemical detection method for the indirect detection of analytes via the direct detection of AgNP labels. A proof-of-concept experiment is shown where streptavidin-coated magnetic microbeads and biotin-coated AgNPs are used to form a composite model analyte. The paper device, called oSlip, and electrochemical method used are easily coupled so the resulting sensor has a simple user-device interface. LODs of 767 fM are achieved while retaining high reproducibility and efficiency. The fourth device is the updated version of the oSlip. In this case, the objective is to show the current progress and limitations in the detection of real analytes using the oSlip device. A sandwich-type immunoassay approach is used to detect human chorionic gonadotrophin (pregnancy hormone) present in human urine. Various optimization steps are performed to obtain the ideal reagent concentrations and incubation time necessary to form the immunocomposite in one step, that is, by mixing all reagents at the same time in the oSlip. Additionally, improvements to the electrochemical detection step are demonstrated.
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Swift recognition of disease-causing pathogens at the point-of-care enables life-saving treatment and infection control. However, current rapid diagnostic devices often fail to detect the low concentrations of pathogens present in the early stages of infection, causing delayed and even incorrect treatments. Rapid diagnostics that require multiple steps and/or elevated temperatures to perform have a number of barriers to use at the point-of-care and in the field, and despite efforts to simplify these platforms for ease of use, many still require diagnostic-specific training for the healthcare professionals who use them. Most nucleic acid amplification assays require hours to perform in a sterile laboratory setting that may be still more hours from a patient’s bedside or not at all feasible for transport in remote or low-resourced areas. The cold-chain storage of reagents, multistep sample preparation, and costly instrumentation required to analyze samples has prohibited many nucleic acid detection and antibody-based assays from reaching the point-of-care. There remains a critical need to bring rapid and accessible pathogen identification technologies that determine disease status and ensure effective treatment out of the laboratory.

Paper-based diagnostics have emerged as a portable platform for antigen and nucleic acid detection of pathogens but are often limited by their imperfect control of reagent incubation, multiple complex steps, and inconsistent false positive results. Here, I have developed mechanisms to economically improve thermal incubations, automate dried reagent flow for multistep assays, and specifically detect pathogenic antigens while improving final output sensitivity on paper-based devices. First, I characterize miniaturized inkjet printed joule-heaters (microheaters) that enable thermal control for pathogen lysis and nucleic acid amplification incubation on a low-cost paper-based device. Next, I explore 2-Dimensional Paper Networks as a means to automate multistep visual enhancement reactions with dried reagents to increase the sensitivity and readability of nucleic acid detection with paper-based devices. Lastly, I aim to create a novel Reverse-Transcription Recombinase Polymerase Reaction mechanism to amplify and detect a specific region of the Spike protein domain of SARS-CoV-2. This will allow the rapid detection of SARS-CoV-2 infections to aid in managing the current COVID-19 pandemic. In the future, these tools could be integrated into a rapid diagnostic test for SARS-CoV-2 and other pathogens, ultimately improving the accessibility and sensitivity of rapid diagnostics on multiple fronts.