Smartphone-integrated paper-based biosensor for sensitive fluorometric ethanol quantification.

The development of fluorometric paper-based analytical devices (fPADs) integrated with smartphone for fluorometric quantification of ethanol in an instrument-free and portable setup is described. The NAD+-dependent alcohol dehydrogenase immobilized within chitosan modified paper substate was utilized as a bio-recognition element and enzymatically generated NADH was used as a fluorescent probe. 3D-printed imaging setup which houses a paper chip holder and UV-light emitting device (LED) was developed for rapid, accurate capture of the fluorescent images. The biocompatible chitosan layer covering the paper provides a feasible environment for enzyme immobilization and enhances the fluorescence signal. The developed fPADs exhibited high sensitivity for ethanol detection and has a linear range for ethanol detection from 17 µM to 8.75 mM (R2 =0.99). Additionally, the fPADs were applied to quantify ethanol in four different wine samples including red, white, rose, and sparkling wines successfully. Moreover, the fPADs produce reproducible signals without loss of enzyme activity for at least 14 days and ~80% activity remained till 28 days. Thus, the proposed approach can provide a facile, affordable, portable, and instrument-free tool for the onsite quantification of ethanol in real samples and is applicable for food quality controls.

Semi-enclosed paper sensor for highly sensitive and selective detection of proline.

In the current study, we have utilized semi-enclosed, leak-proof, microfluidic paper-based analytical devices (µPAD's) modified with isatin conjugated chitosan as specific colorimetric reagent for the detection of proline. Proline is one of the globally accepted stress biomarker in plants and also one of the prominent amino acid present in wine and some processed food. Quantification of proline is regularly required in agriculture field, food and wine industries. Specific interaction of isatin with proline, uniform film forming ability of chitosan which results in uniform coloration and the presence of leak-proof layer which prevent the diffusion of colorimetric reagent deeper resulted in enhancement of color signal intensity at the reaction zone were utilized. Further, the images of the µPAD's were captured using smartphone with 3D printed imaging box which houses smartphone and µPAD's. This platform utilizes smartphone flash for uniform illumination and ensures constant positioning of µPAD's to capture images. This greatly enhances the sensitivity and accuracy of our platform. Compared to previously published highly sensitive multi-layer, paper-based platform for detection of proline, current method has enhanced detection range (∼7 fold) and has comparable limit of detection of 23.75 µM. Moreover, the developed µPAD's platform has reduced optimum reaction temperature and time compared to previous work. The developed paper based platform was utilized for evaluation of proline content in young Arabidopsis plants which are subjected to water stress for 5 days. The devised paper-based methods have the potential to be applicable for the on-site evaluation of various stresses in plants.

In Vitro Blood-Brain Barrier-Integrated Neurological Disorder Models Using a Microfluidic Device.

The blood-brain barrier (BBB) plays critical role in the human physiological system such as protection of the central nervous system (CNS) from external materials in the blood vessel, including toxicants and drugs for several neurological disorders, a critical type of human disease. Therefore, suitable in vitro BBB models with fluidic flow to mimic the shear stress and supply of nutrients have been developed. Neurological disorder has also been investigated for developing realistic models that allow advance fundamental and translational research and effective therapeutic strategy design. Here, we discuss introduction of the blood-brain barrier in neurological disorder models by leveraging a recently developed microfluidic system and human organ-on-a-chip system. Such models could provide an effective drug screening platform and facilitate personalized therapy of several neurological diseases.

Magnetic-Assisted Cell Alignment within a Magnetic Nanoparticle-Decorated Reduced Graphene Oxide/Collagen 3D Nanocomposite Hydrogel.

Hydrogel scaffolds are particularly interesting for applications in tissue engineering because of their ability to create a favorable environment which mimics in vivo conditions. However, the hierarchically ordered anisotropic structure which is found in many native tissues and cellular components is hard to achieve in 3D scaffolds. In this work, we report the incorporation of magnetic nanoparticle-decorated reduced graphene oxide (m-rGO) within a collagen hydrogel. This magneto-responsive m-rGO aligned within the collagen hydrogel during gelation with the application of a low external magnetic field. This nanocomposite hydrogel with magnetically aligned m-rGO flakes is capable of encapsulating neuroblastoma cells (SH-SY5Y), promoting cell differentiation and inducing oriented cell growth owing to its excellent biocompatibility and electrical conductivity. The directionally oriented and differentiated SH-SY5Y cells within the m-rGO collagen hydrogel showed propagation of calcium signal along the direction of orientation. This method can be applied to creating magnetically responsive materials with potential for various biomedical applications.

Recent advances in microfluidic paper-based electrochemiluminescence analytical devices for point-of-care testing applications.

Electrogenerated chemiluminescence (ECL) is an effective method for detecting a wide range of analytes including metal ions, virulent DNA, pathogenic bacteria, tumor cells and glucose. The attractive features of paper including passive liquid transport and biocompatibility are the main two advantages of using paper as a biosensing platform. To achieve key factors in paper-based sensors, the fabrication procedures and the analysis methods are fine tuned to satisfy the requirements of the ultimate-users. Here, we review various ECL signal amplification labels, inexpensive and portable devices, such as rechargeable batteries, which have replaced traditional instrumentation and different light detection technologies used in paper ECL devices. We also highlight the current trends and developments in ECL paper-based microfluidic analytical devices, as well as recent applications of ECL-based detection methods and inexpensive microfluidic devices. We discuss various paper-based devices, including 3D-origami devices, and devices utilizing self-powered and bipolar electrodes. Significant efforts have also been dedicated towards paper based multiplexing analysis (multi-label, and the multi-analyte strategies) and integration of microfluidic lab-on-paper devices with competences for point-to-care diagnostics. This review finally tabulates systematized data on figures of merit and novel types of ECL labels, used for detection of various biomarkers and analytes.

Thin films of silk fibroin and its blend with chitosan strongly promote biofilm growth of Synechococcus sp. BDU 140432.

The activating role of different polymer thin films coated over polystyrene support on the Synechococcus sp. biofilm growth was examined concurrently by measuring biofilm florescence using a dye and by measuring cell density in the isolated biofilm. Compared to blank (no coating), the increase in biofilm formation (%) on silk, chitosan, silk-chitosan (3:2) blend, polyaniline, osmium, and Nafion films were 27.73 (31.16), 21.55 (23.74), 37.21 (38.34), 5.35 (8.96), 6.70 (6.55) and (nil), respectively with corresponding cell density (%) shown in the parentheses. This trend of biofilm formation on the films did not significantly vary for Escherichia coli and Lactobacillus plantarum strains. The films of 20 residues long each of glycine-alanine repeat peptide, which mimics a silk fibroin motif, and a hydrophobic glycine-valine repeat peptide, increased the biofilm growth by 13.53 % and 26.08 %, respectively. Silk and blend films showed highest adhesion unit (0.48-0.49), adhesion rate ((4.2-4.8)×10(-6), m/s) and Gibbs energy of adhesion (-8.5 to -8.6kT) with Synechococcus sp. The results confirmed interplay of electrostatic and hydrophobic interaction between cell-surface and polymer films for promoting rapid biofilm growth. This study established that the thin films of silk and the blend (3:2) promote rapid biofilm growth for all the tested microorganisms.

Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications.

Human serum albumin (HSA)-stabilized Au18 nanoclusters (AuNCs) were synthesized and chemically immobilized on an Indium tin oxide (ITO) plate. The assembly process was characterized by advanced electrochemical and spectroscopic techniques. The bare ITO electrode generated three irreversible oxidation peaks, whereas the HSA-AuNC-modified electrode produced a pair of redox peaks for bilirubin at a formal potential of 0.27V (vs. Ag/AgCl). However, the native HSA protein immobilized on the ITO electrode failed to produce any redox peak for bilirubin. The results indicate that the AuNCs present in HSA act as electron transfer bridge between bilirubin and the ITO plate. Docking studies of AuNC with HSA revealed that the best docked structure of the nanocluster is located around the vicinity of the bilirubin binding site, with an orientation that allows specific oxidation. When the HSA-AuNC-modified electrode was employed for the detection of bilirubin using chronoamperometry at 0.3V (vs. Ag/AgCl), a steady-state current response against bilirubin in the range of 0.2µM to 7µM, with a sensitivity of 0.34µAµM(-1) and limit of detection of 86.32nM at S/N 3, was obtained. The bioelectrode was successfully applied to measure the bilirubin content in spiked serum samples. The results indicate the feasibility of using HSA-AuNC as a biorecognition element for the detection of serum bilirubin levels using an electrochemical technique.

Alcohol oxidase protein mediated in-situ synthesized and stabilized gold nanoparticles for developing amperometric alcohol biosensor.

A simple one step method for the alcohol oxidases (AOx) protein mediated synthesis of gold nano-particles (AuNPs) in alkaline (pH 8.5) condition with simultaneous stabilization of the nanoparticles on the AOx protein surface under native environment has been developed. The formation of the AOx conjugated AuNPs was confirmed by advanced analytical and spectroscopic techniques. The significant increase in zeta potential (ζ) value of -57mV for the synthesized AOx-AuNPs conjugate from the AOx (pI 4.5) protein (ζ, -30mV) implied good stability of the in-situ synthesized nano-conjugate. The AOx-AuNPs conjugate showed steady stability in alkaline (upto pH 8.5) and NaCl (up to 10(-1)M) solutions. The efficiency (Kcat/Km) of the AuNP conjugated AOx was increased by 18% from the free enzyme confirming the activating role of the surface stabilized AuNPs for the enzyme. The AuNPs-AOx conjugate was encapsulated with polyaniline (PANI) synthesized by oxidative polymerization of aniline using H2O2 generated in-situ from the AOx catalysed oxidation of alcohol. The PANI encapsulated AuNPs-AOx assembly was stabilized on a glassy carbon electrode (GCE) by chitosan-Nafion mixture and then utilized the fabricated bioelectrode for detection of alcohol amperometrically using H2O2 as redox indicator at +0.6V. The constructed biosensor showed high operational stability (6.3% loss after 25 measurements), wide linear detection range of 10µM-4.7mM (R(2)=0.9731), high sensitivity of 68.3±0.35µAmM(-1) and low detection limit of 7±0.027µM for ethanol. The fabricated bioelectrode was successfully used for the selective determination of alcohol in beverage samples.

Selective and sensitive detection of free bilirubin in blood serum using human serum albumin stabilized gold nanoclusters as fluorometric and colorimetric probe.

We report here a fluorescence quenching based non-enzymatic method for sensitive and reliable detection of free bilirubin in blood serum samples using human serum albumin (HSA) stabilized gold nanoclusters (HSA-AuNCs) as fluorescent probe. The fluorescence of the nanoclusters was strongly quenched by bilirubin in a concentration dependent manner by virtue of the inherent specific interaction between bilirubin and HSA. A strong binding constant of 0.55×10(6) L mole(-1) between the HSA-AuNC and bilirubin was discerned. The nano clusters each with size ~1.0 nm (in diameter) and a core of Au18 were homogeneously distributed in HSA molecules as revealed from the respective high resolution transmission electron microscopic and mass spectroscopic studies. The fluorescence quenching phenomena which obeyed a simple static quenching mechanism, was utilized for interference free detection of bilirubin with minimum detection limit (DL) of 248±12 nM (S/N=3). The fluorescence response of HSA-AuNCs against bilirubin was practically unaltered over a wide pH (6-9) and temperature (25-50 °C) range. Additionally, peroxidase-like catalytic activity of these nanoclusters was exploited for colorimetric detection of bilirubin in serum sample with a DL of 200±19 nM by following the decrease in absorbance (at λ440 nm) of the reaction and its rate constant (Kp) of 2.57±0.63 mL µg(-1) min(-1). Both these fluorometric and colorimetric methods have been successfully used for detection of free bilirubin in blood serum samples.

A novel amperometric alcohol biosensor developed in a 3rd generation bioelectrode platform using peroxidase coupled ferrocene activated alcohol oxidase as biorecognition system.

Alcohol oxidase (AOx) with a two-fold increase in efficiency (Kcat/Km) was achieved by physical entrapment of the activator ferrocene in the protein matrix through a simple microwave based partial unfolding technique and was used to develop a 3rd generation biosensor for improved detection of alcohol in liquid samples. The ferrocene molecules were stably entrapped in the AOx protein matrix in a molar ratio of ~3:1 through electrostatic interaction with the Trp residues involved in the functional activity of the enzyme as demonstrated by advanced analytical techniques. The sensor was fabricated by immobilizing ferrocene entrapped alcohol oxidase (FcAOx) and sol-gel chitosan film coated horseradish peroxidase (HRP) on a multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode through layer-by-layer technique. The bioelectrode reactions involved the formation of H2O2 by FcAOx biocatalysis of substrate alcohol followed by HRP-catalyzed reduction of the liberated H2O2 through MWCNT supported direct electron transfer mechanism. The amperometric biosensor exhibited a linear response to alcohol in the range of 5.0 × 10(-6) to 30 × 10(-4)mol L(-1) with a detection limit of 2.3 × 10(-6) mol L(-1), and a sensitivity of 150 µA mM(-1) cm(-2). The biosensor response was steady for 28 successive measurements completed in a period of 5h and retained ~90% of the original response even after four weeks when stored at 4 °C. The biosensor was successfully applied for the determination of alcohol in commercial samples and its performance was validated by comparing with the data obtained by GC analyses of the samples.