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E. coli and Salmonella are two of the most common bacterial pathogens involved in food- and water-borne-related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFUmL1 of E. coli in water within 6 hours by targeting the bacterias surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants. Ministry of Education (MOE) Accepted version This work was funded by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-1-025) and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14.

A flow-through colorimetric assay for detection of nucleic acids in plasma is reported. The proposed assay features an array of four polyvinylidene fluoride (PVDF) membranes impregnated with cationic poly (3-alkoxy-4-methylthiophene) (PT) as an optical reporter. The sensing strategy is based on monitoring the changes in optical properties of PT, upon complexation with target nucleic acids in the presence and in the absence of their corresponding complementary peptide nucleic acids (PNAs). As a proof of concept, the proposed methodology is validated using two biomarkers; lung cancer associated microRNA (mir21) and hepatitis B virus DNA (HBV-DNA). The flow-through colorimetric assay enabled detection of mir21 and HBV-DNA in plasma without requiring tedious sample pre-treatment and clean up protocols. Colorimetric responses for mir21 and HBV-DNA were obtained at nanomolar concentrations over five orders of magnitudes (from 1 nM to 10 μM), with a limit of detection of ∼0.6 nM and ∼2 nM in DI water and plasma, respectively. A logic gate system was developed to utilize the colorimetric assay responses as inputs for discrimination of mir21 and HBV-DNA and subsequently to obtain a profile of nucleic acids in samples that exceed respective clinical threshold limits, thereby enabling rapid and point of care (POC) disease diagnosis. Furthermore, the proposed methodology can be utilized for detection of a large number of nucleic acids in plasma by extending the array of PT impregnated membranes incorporated with their corresponding complementary PNAs. Accepted version

Lateral flow assays (LFA) have been extensively explored for rapid and cost effective point-of-care diagnostics. Typically, LFA responses are influenced by the complexity of sample matrices containing analogues or molecules that may potentially yield non-specific responses, for instance, when assaying for biomarkers in blood, serum and plasma. Therefore, isolation of analytes of interest from sample matrices would significantly improve the LFA responses. Herein, we report a magnetic field assisted preconcentration approach for extraction and assay of proteins in an LFA format. Cardiac marker, Troponin (cTnICT complex), is utilized as a model system for validation of the proposed approach. Magnetic fields of different strengths are evaluated for retaining cTnICT on an LFA membrane utilizing magnetic beads conjugated with anti-Troponin I (anti-TnI) antibodies. The sample matrix components subsequently flows through to the absorbent pad via a hydrophilic passivation layer that is protecting the capturing anti-Troponin C (anti-TnC) antibodies in the test zone. Isolated TnI-magnetic bead complexes are then released to flow downstream along the LFA strip to the test zone upon removal of magnetic field and the passivation layer. The capture of cTnICT magnetic bead complexes at the test zone produces a characteristic brownish band, enabling concentration dependant visual detection of cTnICT. Experimental results indicate that the assay yields pM level sensitivity within 15 min using very low sample volumes (

This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate releases a colored byproduct upon cleavage by salivary α-amylase. Introduction of maltose as a noncompetitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20–500 μg/mL) with a limit of detection (LOD) of 8 μg/mL (in aqueous solution). The concentrations of substrate and noncompetitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based “strip” assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific, and easy-to-implement for colorimetric detection of salivary α-amylase between 20 and 500 μg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 μg/mL. The two assay formats are benchmarked against the Phadebas test, a state of the art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses toward stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue. Nanyang Technological University Accepted version This work is supported by Provost’s office and Institute for Sports Research (ISR), School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore.

Analogues and interferents present in complex matrices like blood, plasma or serum influence lateral flow assay (LFA) responses, resulting in weak signals which compromise the limit of detection. Herein, we report a gold nanoparticle conjugated magnetic bead (GMB) based LFA approach for extraction and sensitive visual assaying of proteins in plasma. To validate the approach GMB, conjugated with anti-Troponin I antibodies, is used to capture cardiac marker Troponin I-C-T (cTnICT) in plasma that subsequently flows through on LFA membrane via a test zone containing anti-Troponin C antibodies. The capture of cTnICT-GMB complexes at the test zone produces a characteristic brownish band, enabling concentration dependent visual detection of cTnICT. Subsequently, contrast of these bands are enhanced by a nucleation approach, that utilizes the gold nanoparticles on GMB in the test bands as seed materials for growth of more visible gold clusters, improving visual detection limit of the assay to 0.1 ng/mL in plasma within 20 min, using low sample volumes (

Fly movement was tracked through 3-dimensional (3D) space as the fly died, using either reflected visible light, reflected infrared (IR) light, or fly GFP fluorescence. Behaviors measured included centrophobism, negative geotaxis, velocity, and total activity. In addition, frequency of directional heading changes (FDHC) was calculated as a measure of erratic movement. Nine middle-aged flies were tracked as they died during normal aging, and fifteen young flies were tracked as they died from dehydration/starvation stress. Episodes of increased FDHC were observed 0–8 h prior to death for the majority of the flies. FDHC was also increased with age in flies with neuronal expression of a human Abeta42 protein fragment associated with Alzheimer's disease. Finally, green autofluorescence appeared in the eye and body immediately prior to and coincident with death, and fluorescence of GFP targeted to the retina increased immediately prior to and coincident with death. The results suggest the potential utility of FDHC, green autofluorescence, and retinal GFP as markers of neuronal malfunction and imminent death.

A colorimetric Al3+ sensor based on fluorescence recovery of a conjugated copolymer-ATP complex is proposed. An optimized ratio of two polythiophene (PT) monomers is utilized to synthesize copolymer (CP) that yielded maximized colorimetric response for Al3+ in deionized (DI) and tap water. The electrostatic disassembly of CP-ATP upon addition of Al3+ led to an evident visual color change. The lowest concentration of Al3+ for naked eye observation is around 4 μM, which is below the threshold levels in drinking water according to European Economic Community (EEC) standard. Besides, the proposed assay showed a similar response to Al3+ in tap water. The proposed methodology showed selective and sensitive detection for Al3+ in analytically relevant concentration ranges without involving sophisticated instrumentation, illustrating the applicability for on-site drinking water monitoring., MOE (RG 82/12); Provost Office Nanyang Technological University, Singapore

This article reports on carbon nanotube/manganese dioxide (CNT-MnO2) composites as electrochemical tags for non-enzymatic signal amplification in immunosensing. The synthesized CNT-MnO2 composites showed good electrochemical activity, electrical conductivity and stability. The electrochemical signal of CNT-MnO2 composites coated glassy carbon electrode (GCE) increased by nearly two orders of magnitude compared to bare GCE in hydrogen peroxide (H2O2) environment. CNT-MnO2 composite was subsequently validated as electrochemical tags for sensitive detection of α-fetoprotein (AFP), a tumor marker for diagnosing hepatocellular carcinoma. The electrochemical immunosensor demonstrated a linear response on a log-scale for AFP concentrations ranging from 0.2 to 100 ng mL(-1). The limit of detection (LOD) was estimated to be 40 pg mL(-1) (S/N=3) in PBS buffer. Further measurements using AFP spiked plasma samples revealed the applicability of fabricated CNT-MnO2 composites for clinical and diagnostic applications.

This letter demonstrates a biosensing platform for naked eye detection of miRNA, fabricated using a poly(vinylidene fluoride) thin paper impregnated with positively charged poly(3-alkoxy-4-methylthiophene) as luminescent reporters. The miRNA assay is based on the formation of a duplex and a triplex species between the “reporter and miRNA” and “reporter and miRNA-peptide nucleic acid (PNA) hybrid”, which yields two significantly different optical signals, thereby facilitating naked eye detection. This letter illustrates the successful validation of the proposed methodology via a mir21 assay (miRNA sequence associated with lung cancer). Furthermore, this facile platform enables rapid, sensitive, and selective detection of miRNA, at clinically relevant concentration levels as well as single base pair mismatch, without requiring complex and expensive instrumentation.