Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Detection of high-risk HPV 16 genotypes in cervical cancers using isothermal DNA amplification with electrochemical genosensor.

Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/µL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.

Nucleic Acid Amplification Free-QCM-DNA Biosensor for Burkholderia pseudomallei Detection.

Burkholderia pseudomallei is a gram-negative bacterium that causes the infectious disease melioidosis, a disease that can still be fatal despite appropriate treatment. The bacterium contains the gene clusters for the type III secretion system (TTSS), which are essential for its pathogenicity. This gene was often employed for accurate diagnosis through the laborious process of gene amplification. This work intends to develop a quartz crystal microbalance (QCM)-based TTSS gene detection method without gene amplification approaches to simplify the diagnosis process. In this study, it was demonstrated that a 540 bp sequence flanked by BglI restriction sites within the TTSS1 on the B. pseudomallei genome is an effective target for specific detection of the bacteria. After cultivation and genome extraction, the bacteria can be detected by digesting its genome with BglI in which the TTSS1 fragment is detected by a QCM-DNA biosensor, eliminating the need for nucleic acid amplification. A specific probe designed to bind to the TTSSI fragment was utilized as the receptor on the QCM-DNA biosensor which provided the ability to detect the fragment. The limit of detection of the QCM-DNA biosensor was 0.4 µM of the synthetic DNA target oligonucleotide. The system was also capable of specifically detecting the BglI digested-DNA fragment of B. pseudomallei species with significantly higher signal than B. thailandensis. This study provides evidence for an effective QCM-DNA biosensor that can identify B. pseudomallei without the need for nucleic acid amplification.

Electrochemical aptasensor detection of electron transfer flavoprotein subunit beta for leptospirosis diagnosis.

Electron transfer flavoprotein subunit beta (ETFB) of Leptospira interrogans is a biomarker for diagnosing leptospiral infection. Thus, the ETFB-specific nuclease-resistant RNA aptamer ETFB3-63 was developed and used in an electrochemical aptasensor to assay ETFB. Although the majority of reported biosensors detect various genes and antibodies of L. interrogans, this is the first attempt to construct an electrochemical biosensor to detect ETFB protein for the diagnosis of leptospiral infection. The ETFB protein can be detected without any extraction phase. In this assay, a single-stranded DNA probe complementary to the ETFB3-63 sequence was immobilized on a screen-printed carbon electrode (SPCE). The aptamer was then incubated and hybridized with the antisense probe on the SPCE. In the presence of ETFB, the aptamer dissociates from the aptamer/probe complex on the SPCE to bind with the protein. Methylene blue was then added to intercalate with the remaining hybridized aptamers, and its signal was measured using differential pulse voltammetry. The signal arising from the intercalated methylene blue decreased with increasing concentration of ETFB, showing a linear response in the range of 50-500 nM of ETFB and 10 to 109 leptospira cells per mL, respectively. The aptasensor signal was also specific to L. interrogans but not to 12 related bacteria tested. In addition, the aptasensor showed similar performance in detecting ETFB spiked in human serum to that in buffer, indicating that proteins in the serum do not interfere with the assay. Therefore, this assay has great potential to develop into a point-of-care electrochemical device that is accurate, cost-effective, and user-friendly for leptospirosis diagnosis.

A Label-Free Electrochemical Biosensor for Homocysteine Detection Using Molecularly Imprinted Polymer and Nanocomposite-Modified Electrodes.

An essential biomarker for the early detection of cardiovascular diseases is serum homocysteine (Hcy). In this study, a molecularly imprinted polymer (MIP) and nanocomposite were used to create a label-free electrochemical biosensor for reliable Hcy detection. A novel Hcy-specific MIP (Hcy-MIP) was synthesized using methacrylic acid (MAA) in the presence of trimethylolpropane trimethacrylate (TRIM). The Hcy-MIP biosensor was fabricated by overlaying the mixture of Hcy-MIP and the carbon nanotube/chitosan/ionic liquid compound (CNT/CS/IL) nanocomposite on the surface of a screen-printed carbon electrode (SPCE). It showed high sensitivity, with a linear response of 5.0 to 150 µM (R2 of 0.9753) and with a limit of detection (LOD) at 1.2 µM. It demonstrated low cross-reactivity with ascorbic acid, cysteine, and methionine. Recoveries of 91.10-95.83% were achieved when the Hcy-MIP biosensor was used for Hcy at 50-150 µM concentrations. The repeatability and reproducibility of the biosensor at the Hcy concentrations of 5.0 and 150 µM were very good, with coefficients of variation at 2.27-3.50% and 3.42-4.22%, respectively. This novel biosensor offers a new and effective method for Hcy assay compared with the chemiluminescent microparticle immunoassay at the correlation coefficient (R2) of 0.9946.

A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application.

Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose-response curve of the MOG transfected cell lysate coating plate can be 2-9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance.

Biosensors Based on Ion-Sensitive Field-Effect Transistors for HLA and MICA Antibody Detection in Kidney Transplantation.

This work demonstrates the ability of the Ion-Sensitive Field-Effect Transistor (ISFET)-based immunosensor to detect antibodies against the human leukocyte antigen (HLA) and the major histocompatibility complex class-I-related chain A (MICA). The sensing membrane of the ISFET devices was modified and functionalized using an APTES-GA strategy. Surface properties, including wettability, surface thickness, and surface topology, were assessed in each module of the modification process. The optimal concentrations of HLA and MICA proteins for the immobilization were 10 and 50 µg/mL. The dose-response curve showed a detection range of 1.98-40 µg/mL for anti-HLA and 5.17-40 µg/mL for anti-MICA. The analytical precision (%CV) was found to be 10.69% and 8.92% for anti-HLA and -MICA, respectively. Moreover, the electrical signal obtained from the irrelevant antibody was considerably different from that of the specific antibodies, indicating the specific binding of the relevant antibodies without noise interference. The sensitivity and specificity in the experimental setting were established for both antibodies (anti-HLA: sensitivity = 80.00%, specificity = 86.36%; anti-MICA: sensitivity = 86.67%, specificity = 88.89%). Our data reveal the potential of applying the ISFET-based immunosensor to the detection of relevant anti-HLA and -MICA antibodies, especially in the field of kidney transplantation.

A Multianalyte Electrochemical Genosensor for the Detection of High-Risk HPV Genotypes in Oral and Cervical Cancers.

Infection with high-risk human papillomavirus (HPV) is a major risk factor for oral and cervical cancers. Hence, we developed a multianalyte electrochemical DNA biosensor that could be used for both oral and cervical samples to detect the high-risk HPV genotypes 16 and 18. The assay involves the sandwich hybridization of the HPV target to the silica-redox dye reporter probe and capture probe, followed by electrochemical detection. The sensor was found to be highly specific and sensitive, with a detection limit of 22 fM for HPV-16 and 20 fM for HPV-18, between the range of 1 fM and 1 µM. Evaluation with oral and cervical samples showed that the biosensor result was consistent with the nested PCR/gel electrophoresis detection. The biosensor assay could be completed within 90 min. Due to its simplicity, rapidity, and high sensitivity, this biosensor could be used as an alternative method for HPV detection in clinical laboratories as well as for epidemiological studies.

3D printed hydrophobic barriers in a paper-based biosensor for point-of-care detection of dengue virus serotypes.

Paper-based biosensor is one of the most commonly used platforms for point-of-care testing (POCT). Among these platforms, microfluidic paper-based analytical devices (µPADs) have the most versatile designs due to the different hydrophobic barrier patterns and layers of the devices. In addition, µPADs can also be used in combination with other biosensor platforms to improve the performance of the device. Simple and convenient methods for fabricating low-cost and design-adjustable hydrophobic barriers on paper are one of the most challenging aspects for creating µPADs. This work demonstrated a simple technique for using the common polylactic acid (PLA) filament and wax filament to create hydrophobic barriers on paper for µPADs using a commercialized 3D printer. As a proof of concept, the papers with 3D printed PLA barrier were used in combination with a fluidic chip in a prototype biosensor, in which the barrier paper housed four cell-free reactions and the fluidic chip achieved sample delivery to the reactions in the device. Our designed prototype was capable of discriminating dengue virus serotypes based on small nucleotide sequence differences. The proposed combination of 3D-printed barrier paper and fluidic chip provides a versatile platform for rapid prototyping of POCT with possible compatibility with various detection systems.

Formation of double emulsion micro-droplets in a microfluidic device using a partially hydrophilic-hydrophobic surface.

The objective of this paper is to propose a surface modification method for preparing PDMS microfluidic devices with partially hydrophilic-hydrophobic surfaces for generating double emulsion droplets. The device is designed to be easy to use without any complicated preparation process and also to achieve high droplet encapsulation efficiency compared to conventional devices. The key component of this preparation process is the permanent chemical coating for which the Pluronic surfactant is added into the bulk PDMS. The addition of Pluronic surfactant can modify the surface property of PDMS from a fully hydrophobic surface to a partially hydrophilic-hydrophobic surface whose property can be either hydrophilic or hydrophobic depending on the air- or water-treatment condition. In order to control the surface wettability, this microfluidic device with the partially hydrophilic-hydrophobic surface undergoes water treatment by injecting deionized water into the specific microchannels where their surface property changes to hydrophilic. This microfluidic device is tested by generating monodisperse water-in-oil-in-water (w/o/w) double emulsion micro-droplets for which the maximum droplet encapsulation efficiency of 92.4% is achieved with the average outer and inner diameters of 75.0 and 57.7 µm, respectively.

Optimization of 3-aminopropyltriethoxysilane functionalization on silicon nitride surface for biomolecule immobilization.

The 3-aminopropyltriethoxysilane (APTES) is a common method for biomolecule immobilization on silicon and silicon derivatives such as silicon nitride (Si3N4). However, there are many parameters which impact the efficiency of APTES modification such as APTES concentration and reaction time. Thus, various APTES concentrations (0.1%, 0.5%, 1%, 2%, 5%, and 10%) under different reaction times (15, 30, 60 and 120 min) were compared to achieve the optimal APTES modification condition which produced a thin and stable APTES layer on Si3N4 surface. The modified surfaces were characterized by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy and spectroscopic ellipsometry to determine the wetting property, chemical bonding composition and surface thickness, respectively. In addition, biotin was used as a model to determine the effectiveness of APTES modification condition by coupling with glutaraldehyde (GA). The Alexa Flour 488 conjugated streptavidin was performed to visualize the presence of biotin using fluorescence microscopy due to the specifically binding between biotin and streptavidin. The atomic force microscopy (AFM) was utilized to determine the surface topology which was an indicator to demonstrate the agglomeration of APTES molecule. Moreover, ion sensitive field effect transistor (ISFET) was employed as a biosensor model to demonstrate the effect between surface thickness and sensitivity of biosensor. The results show that the APTES thickness is directly correlated to the APTES concentration and reaction time. Since the importance parameter for ISFET measurement is the distance between biomolecule and sensing membrane of ISFET, the thicker APTES layer negatively impacts the sensitivity of ISFET based biosensor because of the ion shielding effect. Therefore, these results would be valuable information for development of Si3N4 biosensor, especially ISFET based biosensor.

A Novel Nucleic Lateral Flow Assay for Screening phaR-Containing Bacillus spp.

Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.

Development of an immunoFET biosensor for the detection of biotinylated PCR product.

ImmunoFET (IMFET) biosensor is a simple platform for the detection of biotinylated products of polymerase chain reaction (PCR). Construction of the IMFET biosensor started with adsorption of 1.5 mg/mL of protein A (PA) onto the insulated gate surface of ISFET for 90 min. Next, the immobilized 1/500 dilution of anti-biotin antibody was adsorbed onto the PA layer for 60 min. The IMFET biosensor was subsequently ready for detection of the biotinylated amplicon. The IMFET biosensor showed highly specific binding to the biotinylated PCR product of the phaE gene of Haloquadratum walsbyi DSM 16854. The phaE gene is a biomarker of polyhydroxyalkanoate (PHA) producers that contain PHA synthase class III. The lowest amount of DNA template of H. walsbyi DSM 16854 that the IMFET biosensor could detect was 125 fg. The IMFET biosensor has a lower amount of detection compared with a DNA lateral flow biosensor from our previous study. The degree of linearity of the biosensor signal was influenced by the concentration of the biotinylated amplicon. The IMFET biosensor also has a short response time (approximately 30 times) to detect the phaE amplicon compared to an agarose gel electrophoresis. The IMFET biosensor is a promising tool for the detection of the biotinylated PCR product, and it can be integrated into a micro total analysis system (µTAS).

A silicon nitride ISFET based immunosensor for Ag85B detection of tuberculosis.

A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 µg ml-1. This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 µg ml-1 without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

Importance of Thr(328) and Thr(369) for functional maintenance of two receptor-binding ß-hairpins of the Bacillus thuringiensis Cry4Ba toxin: Implications for synergistic interactions with Cyt2Aa2.

Bacillus thuringiensis Cry4Ba mosquito-active toxin was previously shown to utilize two critical loop-residues, Tyr(332) and Phe(364) which are respectively located in ß2-ß3 and ß4-ß5 loops, for synergistic interactions with its alternative receptor-Cyt2Aa2. Here, structural analysis of the Cry4Ba-receptor-binding domain revealed that its N-terminal subdomain encompasses ß2-ß3 and ß4-ß5 hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr(328) in ß2 and Thr(369) in ß5. Functional importance of these two side-chains was demonstrated by single-Ala substitutions (T328A and T369A), adversely affecting toxin activity against Aedes aegypti larvae. Unlike toxicity restoration of the inactive E417A/Y455A toxin mutated within another receptor-binding subdomain, defective bioactivity of T328A and T369A mutants cannot be restored by Cyt2Aa2 as also observed for ß2-ß3 (Y332A) and ß4-ß5 (F364A) loop-mutants. ELISA-based analysis further verified a loss in binding of all four bio-inactive mutants (T328A, Y332A, T369A and F364A) to the immobilized Cyt2Aa2. Protein-protein docking suggested that the two critical loop-residues (Tyr(332) and Phe(364)) correspondingly located at ß2-ß3 and ß4-ß5 loops can clearly interact with four counterpart surface-exposed residues of Cyt2Aa2. Altogether, our present data demonstrate structural importance of Thr(328) and Thr(369) toward hydrogen-bonded stabilization of two receptor-binding hairpins (ß2-ß3 and ß4-ß5) for synergistic toxicity of Cry4Ba with Cyt2Aa2.

The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance (LAMP-QCM) biosensor as a real-time measurement of HPV16 DNA.

We have previously developed quartz crystal microbalance biosensor integrated with loop-mediated isothermal amplification (LAMP-QCM) for human papillomavirus (HPV) type58 DNA detection. Infection with HPV, particularly HPV16, remains a serious health problem due to its major risk factor contributing to cervical cancer. In the present study, LAMP-QCM biosensor was evaluated in terms of a quantitative assay for copy number of HPV16 DNA in cervical samples compared to quantitative PCR using TaqMan assay (TaqMan-qPCR). The detection limit of LAMP-QCM was found to be 10 fold more sensitive than TaqMan-qPCR with 100% specificity and 7.6% imprecision. Different plot of HPV16 DNA copy number using Bland-Altman analysis revealed 94% correlation between LAMP-QCM and qPCR. We therefore concluded that the developed LAMP-QCM biosensor provides a possible rapid and sensitive assay for HPV16 DNA quantification in a routine laboratory.

Two-dye based arrayed primer extension for simultaneous multigene detection in lipid metabolism.

BACKGROUND: Cardiovascular disease (CVD) is one of the major causes of death worldwide. Numerous genetic risk factors in lipid metabolism, including mutations of LDLR, APOB, and PCSK9, as well as polymorphisms of CETP and APOE, have been found to associate with CVD. METHODS: In this study, a two-dye based arrayed primer extension (APEX) microarray assay for simultaneous multigene (LDLR, APOB, PCSK9, CETP, and APOE) detection was developed. The DNA templates, originating from 1 DNA sample of known genotype and 7 blind DNA samples, were amplified by uniplex PCR. RESULTS: Optimized conditions for the APEX reaction were determined to include a hybridization temperature of 55°C and a DNA template size of 50-150bp. The total assay including PCR, purification, fragmentation, APEX reaction, and image analysis could be performed in 6h. In total, 48 genotypes were identified among 8 individual DNA samples by APEX analysis. CONCLUSIONS: The data suggest that this APEX microarray offers a robust, fast, and versatile option for screening these genotypes in hypercholesterolemia patients.

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample.

Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called "Cholera". Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250 µm long and 50 µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5' biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50 µg/mL and 1 µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25 pg or 14 nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835 cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method.

Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization.

Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface.

Microfluidic biosensor for cholera toxin detection in fecal samples.

Sample preparation and processing steps are the most critical assay aspects that require our attention in the development of diagnostic devices for analytes present in complex matrices. In the best scenarios, diagnostic devices should use only simple sample processing. We have therefore investigated minimal preparation of stool samples and their effect on our sensitive microfluidic immunosensor for the detection of cholera toxin. This biosensor was previously developed and tested in buffer solutions only, using either fluorescence or electrochemical detection strategies. The microfluidic devices were made from polydimethylsiloxane using soft lithography and silicon templates. Cholera toxin subunit B (CTB)-specific antibodies immobilized onto superparamagnetic beads and ganglioside GM1-containing liposomes were used for CTB recognition in the detection system. Quantification of CTB was tested by spiking it in human stool samples. Here, optimal minimal sample processing steps, including filtration and centrifugation, were optimized using a microtiter plate assay owing to its high-throughput capabilities. Subsequently, it was transferred to the microfluidic systems, enhancing the diagnostic characteristic of the biosensor. It was found that the debris removal obtained through simple centrifugation resulted in an acceptable removal of matrix effects for the fluorescence format, reaching a limit of detection of only 9.0 ng/mL. However, the electron transfer in the electrochemical format was slightly negatively affected (limit of detection of 31.7 ng/mL). Subsequently, cross-reactivity using the heat-labile Escherichia coli toxin was investigated using the electrochemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrated excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices.