Smartphone-powered, ultrasensitive, and selective, portable and stable multi-analyte chemiresistive immunosensing platform with PPY/COOH-MWCNT as bioelectrical transducer: Towards point-of-care TBI diagnosis.

Traumatic Brain Injury, one of the significant causes of mortality and morbidity, affects worldwide and continues to be a diagnostic challenge. The most desirable and partially met clinical need is to simultaneously detect the disease-specific-biomarkers in a broad range of readily available body fluids on a single platform with a rapid, low-cost, ultrasensitive and selective device. Towards this, an array of interdigitated microelectrodes was fabricated on commercially existing low-cost single-side copper cladded printed-circuit-board substrate followed by the bioelectrodes preparation through covalent immobilization of brain injury specific biomarkers on carboxylic functionalized multi-walled carbon nanotubes embedded polypyrrole nanocomposite modified interdigitated microelectrodes. Subsequently, the immunological binding events were transduced as the normalized change in bioelectrode resistance with and without the target analyte via current-voltage analysis. As proof of concept, current-voltage responses were primarily recorded using a conventional probe station, and later, a portable handheld-electronic-readout was developed for the point-of-care application. The data compilation and analysis were carried out using the in-house developed android-based mobile app. Notably, the smartphone powered the readout through a PL-2303 serial connector, avoiding integrating power sources with the readout. Further, this technology can be adapted to other point-of-care biosensing applications.

Label-free, ultrasensitive and rapid detection of FDA-approved TBI specific UCHL1 biomarker in plasma using MWCNT-PPY nanocomposite as bio-electrical transducer: A step closer to point-of-care diagnosis of TBI.

Traumatic Brain Injury (TBI), a major cause of mortality and neurological disability affecting people of all ages worldwide, remains a diagnostic and therapeutic challenge to date. Rapid, ultra-sensitive, selective, and wide-range detection of TBI biomarkers in easily accessible body fluids is an unmet clinical need. Considering this, in this work, we report the design and development of a facile, label-free, highly stable and sensitive, chemi-impedance-based sensing platform for rapid and wide range detection of Ubiquitin-carboxy terminal hydrolase L1 (UCHL1: FDA-approved TBI specific plasma biomarker), using carboxylic functionalized MWCNTs embedded polypyrrole (PPY) nanocomposites (PPY/f-MWCNT). The said nanocomposites were synthesized using chemical oxidative polymerization method. Herein, the functionalized MWCNTs are used as conducting fillers so as to increase the polymer's dielectric constant according to the micro-capacitor model, thereby augmenting both DC electrical conductivity and AC dielectric property of the nanocomposite. The proposed immunosensing platform comprises of PPY/f-MWCNT modified interdigitated microelectrode (IDµEs) array, on which anti-UCHL1-antibodies are immobilized using suitable covalent chemistry. The AC electrical characterization of the nanocomposite modified IDµEs, with and without the antibodies, was performed through generic capacitance vs. frequency (C-F, 1 KHz - 1 MHz) and capacitance vs. applied bias (C-V, 0.1 V-1 V) measurements, using an Agilent B1500A parametric analyzer. The binding event of UCHL1 peptides to anti-UCHL1-antibodies was transduced in terms of normalised changes in parallel capacitance, via the C-F analysis. Further, we have tested the detection efficiency of the said immunoassay against UCHL1 spiked human plasma samples in the concentration range 10 fg/mL - 1 µg/mL. The proposed sensing platform detected UCHL1 in spiked-plasma samples linearly in the range of 10 fg/mL - 1 ng/mL with a sensitivity and LoD of 4.22 ((ΔC/C0)/ng.mL-1)/cm2 and 0.363 fg/mL, respectively. Further, it showed excellent stability (30 weeks), repeatability, reproducibility, selectivity and interference-resistance. The proposed approach is label-free, and if desired, can be used in conjunction with DC measurements, for biosensing applications.

Artificial Intelligence-Based Portable Bioelectronics Platform for SARS-CoV-2 Diagnosis with Multi-nucleotide Probe Assay for Clinical Decisions.

In the context of the recent pandemic, the necessity of inexpensive and easily accessible rapid-test kits is well understood and need not be stressed further. In light of this, we report a multi-nucleotide probe-based diagnosis of SARS-CoV-2 using a bioelectronics platform, comprising low-cost chemiresistive biochips, a portable electronic readout, and an Android application for data acquisition with machine-learning-based decision making. The platform performs the desired diagnosis from standard nasopharyngeal and/or oral swabs (both on extracted and non-extracted RNA samples) without amplifying the viral load. Being a reverse transcription polymerase chain reaction-free hybridization assay, the proposed approach offers inexpensive, fast (time-to-result: ≤ 30 min), and early diagnosis, as opposed to most of the existing SARS-CoV-2 diagnosis protocols recommended by the WHO. For the extracted RNA samples, the assay accounts for 87 and 95.2% test accuracies, using a heuristic approach and a machine-learning-based classification method, respectively. In case of the non-extracted RNA samples, 95.6% decision accuracy is achieved using the heuristic approach, with the machine-learning-based best-fit model producing 100% accuracy. Furthermore, the availability of the handheld readout and the Android application-based simple user interface facilitates easy accessibility and portable applications. Besides, by eliminating viral RNA extraction from samples as a pre-requisite for specific detection, the proposed approach presents itself as an ideal candidate for point-of-care SARS-CoV-2 diagnosis.

Towards point-of-care diagnosis of Alzheimer's disease: Multi-analyte based portable chemiresistive platform for simultaneous detection of ß-amyloid (1-40) and (1-42) in plasma.

Label-free simultaneous detection of Alzheimer's disease (AD) specific biomarkers Aß40 and Aß42 peptides on a single platform using polypyrrole nanoparticle-based chemiresistive biosensors is reported here. The proposed interdigitated-microelectrode based inexpensive multisensor-platform can concurrently detect Aß40 and Aß42 in spiked-plasma in the range of 10-14 - 10-6 g/mL (with LoDs being 5.71 and 9.09 fg/mL, respectively), enabling the estimation of diagnostically significant Aß42/Aß40 ratio. A detailed study has been undertaken here to record the individual sensor responses against spiked-plasma samples with varying amounts and proportions of the two target peptides, towards enabling disease-progression monitoring using the Aß-ratio. As compared to the existing cost-ineffective brain-imaging techniques such as PET and MRI, and the high-risk CSF based invasive AD biomarkers detecting procedures, the proposed approach offers a viable alternative for affordable point-of-care AD diagnostics, with possible usage in performance evaluation of therapeutic drugs. Towards point-of-care applications, the portable readout used in this work was conjugated with an android-based mobile app for data-acquisition and analysis.

Cerium oxide nanofiber based electroanalytical sensor for TNF-α detection: Improved interfacial stability with Nafion.

The present work is aimed at improving the adhesion of nanomaterials at the interface of solid state working electrodes. Towards this, herein, an efficient method has been proposed that requires the electrode interface to be decorated with an optimally thin layer of Nafion. This selectively permeable layer ensures the stability of the sensor interface, without hampering the transport of biomolecules and electrons. As a case study, here, electrospun Cerium oxide nanofiber (CeNF) modified Glassy carbon electrodes (GCE) have been used as the sensing interface, and stability and performance of the GCE/CeNF/Nafion interface is evaluated using analytical electrochemistry. The CeNF is synthesized via electrospinning and is characterized using X-ray diffraction spectroscopy, Thermal gravimetry, Fourier transform infrared spectroscopy, and Field emission scanning electron microscopy. Further, detection of sepsis specific biomarker TNF-α from spiked buffer samples is demonstrated, as a case study, towards evaluating the effect of Nafion on the interfacial sensitivity. The achieved LOD of GCE/CeNF and GCE/CeNF/Nafion for TNF-α detection were 2.8 fg/mL and 1.2 fg/mL, respectively. A comparative analysis between the Electrochemical impedance spectroscopic (EIS) results of the GCE/CeNF and the GCE/CeNF/Nafion interfaces confirms the improvement in stability, without affecting the sensitivity and the limiting detection.

Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis.

This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.

A miniaturized electrochemical platform with an integrated PDMS reservoir for label-free DNA hybridization detection using nanostructured Au electrodes.

We report the fabrication and characterization of a miniaturized electrochemical platform for the label-free detection of DNA hybridization. The proposed platform is fabricated using microfabrication and electrodeposition techniques. Comprising a Ti working electrode with electrodeposited Au nanostructures, and Pt/Au pseudo-reference and counter electrodes, the device accounts for a limit of detection of 0.97 fM and a sensitivity of 20.78 (µA µM-1) cm-2 with respect to Dengue virus specific consensus primer detection in the range of 10 fM-1 µM. Here, the incorporation of nanostructured Au in the active sensing area not only enhances the current response by increasing the overall surface area, but it also facilitates facile probe DNA immobilization by gold-thiol self-assembly. We have used differential pulse voltammetry analysis in this study to monitor the changes in reaction kinetics with respect to target hybridization. Furthermore, the evaluation of reproducibility of the biosensor and its selectivity against interference has yielded acceptable outcomes. Additionally, in order to evaluate the system's selectivity, we have successfully distinguished PCR amplified wild type and mutant target DNAs corresponding to the BRCA1 specific gene. Here, the mutant and the wild type target DNAs differ by a two base deletion, and the fact that the system is able to differentiate even such minute dissimilarities under hybridization conditions is indicative of its superior performance.

Electrospun tin (IV) oxide nanofiber based electrochemical sensor for ultra-sensitive and selective detection of atrazine in water at trace levels.

Atrazine, a class 3a carcinogen, is a pesticide of chloro triazine family and is known to severely affect the human endocrine system upon consumption. The toxic effects of atrazine cause damage not only to the humans but also to animals and plants. In lieu of the detrimental effects of atrazine on environment, it is essential to develop a sensor platform capable of its detection in water. Here, we propose ultrasensitive electrochemical detection of atrazine using electrospun SnO2 nanofibers. In this study, the nanofibers have been characterized using Field Emission Spectroscopy, X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), UV-Vis-NIR spectroscopy and Fourier transform infrared spectroscopy (FTIR). Using a label-free transduction, we have detected atrazine in fairly low concentrations, with the limit of detection being 0.9 zM and the sensitivity being 4.11 (µA/µM)/cm2, in a wide dynamic detection range varying from 1 zM to 1 µM. Furthermore, we have reported atrazine detection in trace levels in spiked real time water samples, which is an essential step in ensuring that the sensing platform can be deployed for practical applications. In addition to this, the sensor exhibits excellent selectivity, reasonable stability (when stored at 4 °C), and good interference-resistance.

Chemiresistive DNA hybridization sensor with electrospun nanofibers: A method to minimize inter-device variability.

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn2O3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM - 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM - 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM.

Recent advances in biosensors for diagnosis and detection of sepsis: A comprehensive review.

Sepsis is one of the leading causes of mortality among critically ill patients globally. According to WHO report 2018, it is estimated to affect beyond 30 million people worldwide every year. It causes loss of human lives, which arise from infection and inflammation and long term stay in intensive care unit (ICU) in hospitals. Despite the availability of satisfactory prognostic markers contributing to the diagnosis of sepsis, millions of people die even after admission to the hospitals. Correct and early diagnosis of sepsis leads to rapid administration of appropriate antibiotics can thus potentially avert the attainment to critical stages of sepsis, thereby saving human lives. Conventional diagnostic practices are costly, time consuming and they lack adequate sensitivity and selectivity, provoking an urgent need for developing alternate sepsis diagnosis systems. Nevertheless, biosensors have the much-treasured scope for reasonable sepsis diagnosis. Advancement in nano-biotechnology has provided new paradigm for biosensor platforms with upgraded features. Here, we provide an overview of the recent advances in biosensors with a brief introduction to sepsis, followed by the conventional methods of diagnosis and bio-sensing. To conclude, a proactive role and an outlook on technologically advanced biosensor platforms are discoursed with possible biomedical applications.

Electrospun manganese (III) oxide nanofiber based electrochemical DNA-nanobiosensor for zeptomolar detection of dengue consensus primer.

Nanoscale biosensors, owing to their high-sensitivity and extremely low limits-of-detection, have enabled the realization of highly complex and sophisticated miniaturized platforms for several important healthcare applications, the most predominant one being disease diagnosis. In particular, nanomaterial facilitated electrochemical detection of DNA hybridization has had an exceptional impact on fields such as genetics and cancerous mutation detection Here we report an ultrasensitive electrochemical platform using electrospun semi-conducting Manganese (III) Oxide (Mn2O3) nanofibers for DNA Hybridization detection. The proposed platform coalesces the inherent advantages of metal-oxide nanofibers and electrochemical transduction techniques, resulting in label-free zeptomolar detection of DNA hybridization. As proof of concept, we demonstrate zeptomolar detection of Dengue consensus primer (limit of detection: 120×10-21M) both in control as well as spiked serum samples. Our reported detection limit is superior in comparison with previously reported electrochemical DNA hybridization sensors for Dengue virus detection, spanning both labeled and label-free transductions. This ultra-sensitivity, we believe, is a result of synthesizing a low bandgap electrospun metal-oxide nanomaterial corresponding to a specific oxidation state of Manganese. This methodology can be extended for detection of any hybridization of interest by simply adapting an appropriate functionalization protocol and thus is very generic in nature.

A comprehensive approach for milk adulteration detection using inherent bio-physical properties as 'Universal Markers': Towards a miniaturized adulteration detection platform.

This paper proposes a novel milk quality detection approach based on utilization of inherent biophysical properties as 'markers' for adulteration. Unlike the traditional adulterant-specific approaches, this method is generic and universal. It exploits the change in innate milk properties, such as electrical conductivity and pH, upon addition of adulterants as a transduction mechanism for detecting milk adulteration. In this work, adulteration with more than 10 commercially known hazardous adulterants is detected by monitoring the changes in milk electrical conductivity and pH. The electrical parameters for pure milk were standardized using AC impedance-spectroscopy with glassy carbon working electrode and platinum counter/reference electrode at a potential of 0.3V and in the frequency range of 1Hz-1MHz. The experiments were repeated using gold-electrodes fabricated on glass-substrate as a first step towards developing a miniaturized platform. The concept of a 'unified-universal-marker' for successful prediction of adulteration is accentuated in this work.

An ultrasensitive label free nanobiosensor platform for the detection of cardiac biomarkers.

We report the fabrication of a label free nano biosensor platform comprising single nanofiber that is derived out of multi-walled carbon nanotubes (MWCNTs) embedded SU-8 photoresist, for the detection of three important human cardiac biomarkers viz., myoglobin (Myo), cardiac Troponin I (cTn I) and Creatine Kinase-MB (CK-MB). These composite nanofibers were synthesized using electrospinning process. Single nanofibers were aligned between pairs of electrodes in-situ during the electrospinning process. The target proteins were detected using chemiresistive detection methodology. Each biomarker was detected using a specific, single, aligned nanofiber, functionalized with its corresponding monoclonal antibody. Chemiresistive detection involves measuring the change in conductance of the functionalized nanofibers upon the binding of the targeted antigen. The minimum detection limits of Myo, CK-MB and cTn I were experimentally found out to be as low as 6, 20 and 50 fg/ml respectively. No response was observed when the nanofibers were exposed to a non-specific protein, demonstrating excellent specificity to the targeted detection. These MWCNTs embedded SU-8 nanofibers based nanobiosensor platform shows great promise in the detection of cardiac markers and other proteins as they have fast response time, high sensitivity and good specificity.

A highly sensitive self assembled monolayer modified copper doped zinc oxide nanofiber interface for detection of Plasmodium falciparum histidine-rich protein-2: Targeted towards rapid, early diagnosis of malaria.

Rapid, ultrasensitive diagnostic/triaging kits for early detection of malarial parasites are critical for prevention of malarial epidemic, especially in developing and tropical countries. Unlike traditional microscopic diagnosis, these kits rely on the detection of antigens specific to malarial parasites. One such antigen which is routinely used in these diagnostic kits is Histidine-rich protein-2; a protein synthesized and released into the blood stream by the parasite Plasmodium falciparum. In this paper, we demonstrate an ultrasensitive nanobiosensor detection platform for Histidine-rich protein-2 having a limit of detection of attogram/ml. This nanobiosensor platform comprises of Mercaptopropylphosphonic acid functionalized copper doped zinc oxide nanofibers synthesized by electrospinning technique. Ultrasensitivity of attogram/ml can be attributed to the complimentary effects of Mercaptopropylphosphonic acid and copper doping in zinc oxide. Mercaptopropylphosphonic acid enhances the functional groups required for immobilizing antibody. Copper doping in zinc oxide not only increases the conductivity of the nanofibers but also pre-concentrates the target analyte onto the Mercaptopropylphosphonic acid treated nanofiber surface due to inherent electric field generated at the copper/zinc oxide heterojunction interface. The impedimetric detection response of copper-doped zinc oxide nanofiber modified electrode shows excellent sensitivity (28.5 kΩ/(gm/ml)/cm(2)) in the detection ranges of 10 ag/ml-10 µg/ml, and a detection limit of 6 attogram/ml. In addition, the proposed biosensor is highly selective to targeted HRP2 protein with a relative standard deviation of 1.9% in the presence of various interference of nonspecific molecules. To the best of our knowledge, this biosensor shows the lowest detection limit of malarial parasites reported in the literature spanning different nanomaterials and different detection mechanisms. Since the nanobiosensor platform is based on immunoassay technique, with a little modification, it can be extended for developing point-of-care diagnostic devices for several biomarkers of importance.