Advanced Materials for Rapid Diagnostics in Food, Agriculture and Healthcare

Paper based devices are an emerging trend for micro total analysis systems due to their rapid, sensitive and specific attributes. Lateral flow assays (LFAs), the predecessor of paper-fluidic devices, are developed for many applications which range from nucleic acid and antibody detection to commercial home pregnancy tests. With growing interest in replacing conventional detection methods, multistep assays are needed. These assays require multiple test reagents, therefore, there is a need to control fluid flow for the development of complex paper-based devices. We were able fabricate paper-fluidic platforms where we used electrowetting-on-dielectrics to create valves on paper. With this method, we directly controlled the timing and flow of the fluid. However, the electrowetting valves required an external power source for actuation, thus we developed a passive fluid control method. To do this, passive delay valves/barriers were inkjet-printed, by altering the drop spacing of the ink creating effective delay barriers, which decreases the rate of capillary action of the throughput solution. The patterned devices were later used to create a paper based device where an amplified nucleic acid assay was performed. After creating a paper based platform and understanding the properties that control fluid flow on paper, it is important to discover applications that are suitable for the devices. Phage amplified LFAs were developed, where a genetically engineered phage that overexpresses alkaline phosphatase was used, in combination with phage amplification kinetics to detect for low levels of E. coli in a sample. In the end, we were able to fabricate a barcode style LFA that had a visually quantitative colorimetric readout. After understanding phage kinetics and amplification, their infectious and destructive mechanism was leveraged and applied to decontamination of agricultural rinse water. In general, bacteriophages are capable of infecting and lysing target bacteria and are kept viable by lyophilization, but freeze-drying is time consuming and requires large machinery. In our study, we dehydrated bacteriophages in electrospun nanofibers and studied the effects of excipients in polymeric solutions and different storage conditions on bacteriophage viability. Ultimately, electrospun nanofibers stored for eight weeks at ambient temperatures retained high phage viability which is sufficient for infection

Investigation of reagent storage and electrochemical testing on filter paper

Thesis (Ph.D.)--Boston UniversityDiagnosis and detection is one of the most effective means of controlling matters that adversely affect public health and safety. Yet, in the developing world with a high burden of disease, most gold standard diagnostics remain widely inaccessible due to cost and lack of infrastructure. In recent years, one strategy to increase access to health and safety devices has been through the development of point-of-care diagnostics that are low-cost, portable, and easy-to-use for on-site analysis. In particular, paper has recently been in the spotlight as such a point-of-care (POC) platform. Compared to conventional POC tests made of glass or plastic substrates, paper itself is even thinner, light-weight, portable, disposable, and can store biological and chemical molecules for analytical measurement within its fibrous network. Several paper-based tests have demonstrated high sensitivity and specificity to detect proteins, bacteria, and metals for applications in disease diagnosis, health monitoring, and food and water safety. However, several gaps still remain in order to fully develop these paper-based analytical devices for point-of-care use in low-resource settings. First, reagent stability on filter paper is poorly understood, as well as its influence on quantitative, long-term testing. Second, the need for specialized instrumentation to perform the analytical methods on the paper devices can be a logistical and financial burden to end users in resource-limited settings. This dissertation addressed these questions through the development of quantitative paper assays for robust and point-of-care testing in low-resource settings. First, we fabricated micro-paper electrochemical devices, or µPEDs, for the amperometric detection of ethanol. This target analyte has direct applications in the global issue of road safety, which claims thousands of lives due to driving under the influence of alcohol. Also, we demonstrate that ethanol detection can provide the basis for the novel detection of substandard misoprostol, a high impact drug to save mothers from post-partum bleeding that is often the reason for maternal mortality. Second, we developed an independent method to study reagent stability on filter paper under conditions likely encountered in low-resource settings. Methods that enhanced stability were also used in the development of the µPEDs. Finally, we demonstrate that the ethanol measurements on our µPEDs could be performed with a commercial glucose meter, which operate by the same principles required to measure analyte concentrations. This integration of device and reader presents a cheap, reliable, low-power, and portable platform that can be adapted for the detection of other analytes relevant to health and safety

Application of Paper-Based Microfluidic Analytical Devices (μPAD) in Forensic and Clinical Toxicology: A Review

The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (μPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, μPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors

Thermoplastic microfluidic technologies for portable and disposable bioanalytical and diagnostic platforms

Portable and cost-effective medical diagnostic technologies that require minimal external infrastructure for their operation are highly desirable for on-field military operations, defense against acts of bioterrorism, and infectious disease screening in resource-limited environments. Miniaturized Total Analysis Systems (µTAS) have the potential to fulfill this un-met need via low-cost, portable, and disposable point-of-care (POC) diagnostic devices. Inherent advantages of µTAS systems can be utilized to transform diagnostic technologies that currently require significant investment in centralized laboratories and highly trained personnel into automated, integrated, and miniaturized platforms. This dissertation addresses the development of microfabrication techniques and resulting component technologies that are realized in low-cost thermoplastic substrates. A thermoplastic microfabrication technique termed orogenic microfabrication, based on a non-reversible solvent-assisted swelling mechanism, is developed to provide unique capabilities for microscale and nanoscale patterning in rigid thermoplastics with minimal infrastructure. Orogenic microfabrication is compatible with multiple masking techniques including photolithography, chemical surface modification, contact and noncontact spotting, and inkjet deposition techniques, with each masking method offering unique influence on resulting orogenic structures that can be applied to microfluidic and µTAS systems. Direct ink masking is further explored as a low-cost rapid prototyping tool for fabrication of simple microfluidic devices where channel formation and bonding are combined into a single step, resulting in fully enclosed microfluidic channels within 30 minutes. Chemical surface passivation by UV-ozone treatment is utilized in combination with orogenic swelling and thermocompression bonding to develop single-use burst valves with tunable burst pressures. In addition to assisting in on-chip fluid manipulation, the normally closed burst valves enable on-chip reagent packaging and hermetic sealing of bioactive material in lyophilized format, and can be used for delivery of stored reagents for a range of disposable point-of-care assays. On-chip integrated micropumps are also developed, using simple fabrication process compatible with conventional thermoplastic fabrication techniques such as direct micromilling or injection molding. Direct displacement of liquid reagents using screw-assisted pumping can be operated either automatically or manually, with on-demand delivery of liquid reagents in a wide range of flow rates typically used in microfluidic applications. Collectively, the technologies developed in this dissertation may be applied to the future development of simple, disposable, and portable diagnostic devices that have the potential to be operated without off-chip instrumentation. On-chip storage of buffers and reagents in either dry or liquid format, and on-demand delivery of liquid reagents is packaged in a miniaturized, portable, and automated platform that can be operated in resource-constrained settings by practitioners with minimal expertise

Application of Paper-Based Microfluidic Analytical Devices (µPAD) in Forensic and Clinical Toxicology: A Review

The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (μPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, μPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma- hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors

Bioanalytical applications of microfluidic devices

The first part of the thesis describes a new patterning technique--microfluidic contact printing--that combines several of the desirable aspects of microcontact printing and microfluidic patterning and addresses some of their important limitations through the integration of a track-etched polycarbonate (PCTE) membrane. Using this technique, biomolecules (e.g., peptides, polysaccharides, and proteins) were printed in high fidelity on a receptor modified polyacrylamide hydrogel substrate. The patterns obtained can be controlled through modifications of channel design and secondary programming via selective membrane wetting. The protocols support the printing of multiple reagents without registration steps and fast recycle times. The second part describes a non-enzymatic, isothermal method to discriminate single nucleotide polymorphisms (SNPs). SNP discrimination using alkaline dehybridization has long been neglected because the pH range in which thermodynamic discrimination can be done is quite narrow. We found, however, that SNPs can be discriminated by the kinetic differences exhibited in the dehybridization of PM and MM DNA duplexes in an alkaline solution using fluorescence microscopy. We combined this method with multifunctional encoded hydrogel particle array (fabricated by stop-flow lithography) to achieve fast kinetics and high versatility. This approach may serve as an effective alternative to temperature-based method for analyzing unamplified genomic DNA in point-of-care diagnostic

Produção e caracterização da fusão ZZapo-CBM64 para captura e deteção da apolipoproteína-A1 em testes de papel

Mestrado em Bioquímica - Métodos BiomolecularesAffibodies (ZZ) são pequenas proteínas de afinidade que podem ser modificadas para se ligarem a alvos específicos. Estas proteínas têm sido sugeridas como uma alternativa a anticorpos, devido ao seu processo de produção mais simples e barato. Assim, o objetivo deste trabalho de mestrado visou a possibilidade da utilização de um affibody (ZZapo) para a captura da Apolipoproteina-A1 (Apo-A1) e sua deteção com anticorpos Anti-Apo-A1 conjugados com Nanopartículas de Ouro (AuNPs) num Dispositivo Microfluídico Analítico de Papel (μPAD). Para atingir o objetivo proposto, a fusão de uma molécula com afinidade a carboidratos da família 64 (CBM64) com o affibody ZZapo (ZZapo-CBM64) foi desenhada, produzida em E.coli, sequenciada, purificada e quantificada com sucesso. A fusão ZZapo-CBM64 foi comparada com a fusão semelhante ZZ-CBM64, que tem afinidade para imunoglobulina G (IgG). Foi testada a capacidade de ligação das fusões à celulose, tendo-se verificado que ambas apresentaram alta afinidade a micropartículas e papel de celulose. Foi ainda testada a capacidade de ligação das duas fusões a IgG, pela utilização de IgG marcado com fluorescência. No entanto, ao contrário da ZZ-CBM64, a ZZapo-CBM64 não mostrou afinidade para a IgG. Um teste μPAD foi criado com barreiras impressas a cera hidrofóbica, e com um adesivo na base do teste, o que permitiu os testes serem feitos em superfícies planas e ainda contribuiu para um fluxo da amostra mais rápido. Os testes μPAD mostraram interações não específicas entre ZZapo-CBM64 e AuNPs, que foram removidas pela conjugação das AuNPs com Albumina de Soro Bovino (BSA) e adição de BSA e Tween20 à solução tampão. Os testes μPAD para deteção da Apo-A1 mostraram interações não específicas entre Apo-A1 e a membrana adesiva, que foram removidas pela utilização de um tampão de Bicarbonato de Amónia com BSA e Tween20. A deteção de Apo-A1 em μPADs não foi conseguida devido ao ZZapo-CBM64 não capturar a Apo-A1. Esta falha poderá ser devido a algum bloqueio da zona de captura relacionado com a estrutura 3D da fusão. De modo a ultrapassar esta questão, deverão ser realizados trabalhos futuros para o estudo da estrutura 3D da fusão, assim como estudo de outras fusões com diferentes variantes de affibodies para averiguar se o problema é exclusivo à fusão ZZapo-CBM64 ou não.Affibodies (ZZ) are small affinity proteins that can be engineered to bind to specific targets. These molecules have emerged as an alternative to antibodies due to their simpler and cheaper production process. The objective of this work was thus to assess the possibility of using an affibody (ZZapo) to capture Apolipoprotein-A1 (Apo-A1) and its detection using Anti-Apo-A1 antibodies conjugated to Gold Nanoparticles (AuNPs) on a Microfluidic Paper-Based Analytical Device (μPAD). To achieve the proposed objective, a fusion of a Carbohydrate Binding Molecule of the family 64 (CBM64) with a ZZapo affibody (ZZapo-CBM64) was successfully designed, produced in E. coli, sequenced, purified and quantified. The ZZapo-CBM64 fusion was then compared to a similar ZZ-CBM64 fusion (produced and purified in the same way) with immunoglobulin G (IgG) binding capacity, and tested for its cellulose binding capacity. Both fusions showed high affinity to cellulose particles and paper. They were also tested for IgG binding capacity, using a fluorescently labelled IgG. While ZZ-CBM64 successfully captured IgG, the new ZZapo-CBM64 did not capture the labelled IgG. A μPAD test was designed and produced with wax printed hydrophobic barriers, and the use of an adhesive membrane in the bottom of the test enabled running tests on a flat surface, and contributed for faster sample flow. μPAD tests showed that ZZapo-CBM64 and AuNPs had non-specific interactions, which were removed by conjugating AuNPs with Bovine Serum Albumin (BSA) and using a buffer containing BSA and Tween20. μPAD tests for the detection of Apo-A1 showed non-specific binding of Apo-A1 and the adhesive membrane, which was removed by using ammonium bicarbonate buffer. Apo-A1 detection in μPADs was unsuccessful, which was shown to be caused by ZZapo-CBM64 failing to capture Apo-A1. This failed capture could be caused by the capture zone of the protein being blocked. Future works should be directed to the study of the 3D structure of this fusion, as well as the study of other fusions with different affibody variants to assess if this problem is exclusive to ZZapo-CBM64 or not

Genipin cross-linked chitosan for signal enhancement in the colorimetric detection of aflatoxin B1 on 3MM chromatography paper

Detection of mycotoxins by conventional methods such as ELISA or LC-MS can be expensive and time-consuming. Therefore, paper-based biosensors can be effectively used for on-site analysis, due to their low cost and easy detection procedures. Nevertheless, even when the application of colorimetric methods on paper enhance the simplicity and affordability of multiple determinations, the signal intensity and final readout can be affected by a limited color uniformity. In this work, Ellman’s method for the quantification of aflatoxin B1 was utilized as a model colorimetric assay on paper, in which the test zones were modified with chitosan-immobilized enzyme (AChE). A comparison of the cross-linking effect of genipin on two chitosans of varying molar mass and degree of acetylation, exhibited a greater signal enhancement from the sample with a higher degree of acetylation and molecular weight

Huokoisten materiaalien hydrofobinen kuviointi mustesuihkutulostuksella mikrofluidistisia sovelluksia varten

Paperfluidic devices are microfluidic devices patterned out of paper or other highly porous material. Liquid transport in paperfluidic devices is propelled either by surface wetting or interior capillary wicking. The direction of aqueous liquid flow on such devices is controlled by selective patterning of hydrophobic barriers on an otherwise hydrophilic base substrate. A variety of hydrophobic materials and printing methods have been lately demonstrated as feasible for producing such patterns. Unlike conventional graphic printing, hydrophobising ink has to penetrate the whole depth of the wicking substrate, in order to produce properly functioning leak-free barriers. One major expected application area for paperfluidics in the future is in the field of lab-on-a-chip devices, intended to provide simple, transportable, disposable and self-sufficient analysis tools for medical diagnostics and environmental monitoring. On such devices, fluid samples flow along hydrophilic channels through assay zones, where they chemically interact with pre-applied reagents. This study focused on the development of simple solvent-based hydrophobic inks for inkjet printing of microfluidic patterning on paper substrates. Hydrophobic inks were produced by dissolving alkyl ketene dimer (AKD), paraffin wax and low molecular weight polystyrene in p-xylene. Hydrophobic test patterns were created by inkjet printing these inks on two highly porous filter papers. AKD ink was found to produce effective hydrophobic barriers but with poorly defined borders. Polystyrene ink produced well defined borders, but could only penetrate the full depth of the substrate on one paper. Channels 680 ± 80 μm wide and barriers 883 ± 91 μm wide could be produced with it. Adding polystyrene as a rheological modifier to AKD ink improved jetting frequency. Paraffin wax could not be dissolved in sufficient amounts to produce proper barriers. Hydrophobic ink penetration into filter paper was found to take place as film flow rather than through complete filling of pores. Paper properties and ink viscosity were considered to play a role in providing ink with a quick access to the reverse side. Differences in border definition might be due to the well-known coffee stain effect and different interaction with the cellulose fibre surfaces.Paperifluidiset laitteet ovat mikrofluidisia laitteita, jotka muodostetaan hydrofobisesti kuvioimalla paperia tai muuta huokoista materiaalia. Tällaisissa laitteissa vesi virtaa spontaanisti pintajännityksen ja kapillaaripaineen vaikutuksesta samalla kun hydrofobiset seinämät ohjaavat virtausta hydrofiilisia kanavia pitkin. Virtauskuvioita voidaan valmistaa olemassa olevilla painatusmenetelmillä käyttämällä hydrofobista materiaalia sisältäviä musteita. Toisin kuin perinteisessä graafisessa painatuksessa, hydrofobisia virtauskuvioita valmistettaessa musteen täytyy tunkeutua paperiin koko sen syvyydeltä muodostaakseen toimivia seinämiä. Tulevaisuudessa paperifluidistiikan odotetaan tarjoavan alustan yksinkertaisille, helposti kuljetettaville ja kertakäyttöisille testilaitteille muun muassa lääketieteellisessä diagnostiikassa ja ympäristön laadun tarkkailussa. Tällaisissa laitteissa nestemäiset näytteet virtaavat hydrofiilisia kanavia pitkin testialueille, joissa ne reagoivat valmistusvaiheessa lisättyjen kemikaalien kanssa. Tässä tutkimuksessa keskityttiin tutkimaan yksinkertaisten liuotinpohjaisten mustesuihkumusteiden käyttöä virtauskuvioiden muodostamiseen paperille. Hydrofobiset musteet valmistettiin liuottamalla alkyyliketeenidimeeriä (AKD), parafiinia ja pienimolekyylimassaista polystyreenia p-ksyleeniin. Virtauskuviot muodostettiin tulostamalla musteita kahdelle eri suodatinpaperille. AKD-pohjainen muste muodosti tehokkaita hydrofobisia esteitä, tosin esteiden reuna-alueet olivat epätarkkoja. Polystyreeni-pohjainen muste muodosti tarkkarajaisia kuvioita, mutta tunkeutui riittävästi paperin läpi vain toisella testatulla paperilla. Sillä voitiin kuitenkin valmistaa 680 ± 80 μm leveitä kanavia ja 883 ± 91 μm leveitä hydrofobisia seinämiä. Lisäksi pienen polystyreenimäärän lisääminen AKD-pohjaiseen musteeseen paransi syntyneen musteen tulostustaajuutta. Parafiini ei liuennut riittävissä määrin muodostaakseen tehokkaita esteitä paperille. Hydrofobisten musteiden tunkeutuminen paperiin tapahtui pääasiallisesti pintavirtauksena kuitujen pintoja pitkin, näiden välisten huokosten täyttymisen sijaan. Paperin ominaisuudet ja musteen viskositeetti olivat merkittävässä osassa musteen tunkeutumissyvyyden kannalta. Erot hydrofobisten kuvioiden reuna-alueiden tarkkuudessa saattavat johtua hyvin tunnetusta kahvitahrailmiöstä ja hydrofobisoivien materiaalien erilaisesta vuorovaikutuksesta selluloosakuidun pinnan kanssa