Colorimetric and Fluorometric Profiling of Advanced Glycation End Products.

Profiling of advanced glycation end products (AGEs) is an emerging area of clinical significance for disease diagnosis and prognosis. Typically, concentrations of AGEs are estimated in laboratories by trained personnel using sophisticated equipment. Herein, a facile approach for colorimetric and fluorometric profiling of AGEs is reported for rapid and on-site analysis. The concentrations of AGE levels in plasma are estimated via changes in optical properties of polythiophenes (PTs) upon interaction with aptamers (Apts) in the presence and in the absence of AGEs. To validate the proposed approach, glyceraldehyde-derived AGEs (AGE class 1 [AGE1]), the biomarker associated with cardiovascular diseases and diabetes, are used as a model system. Colorimetric analysis yielded linear responses for AGE1 for clinically relevant concentration ranges between 1.5 and 300 µg/mL with a limit of detection (LOD) of ∼1.3 µg/mL. Subsequently, an approach utilizing PTs with four different pendant groups in conjunction with four different Apts is demonstrated for qualitative colorimetric profiling and for quantitative fluorometric profiling of up to four AGEs in clinical matrices. Principal component analysis (PCA) of fluorometric responses of AGE-spiked samples yielded distinct responses for the different AGEs tested. Thus, the proposed approach ascertains rapid profiling of spiked AGEs in plasma samples without the requirement of preanalytical processing and advanced instrumentation, thereby facilitating on-site diagnosis.

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne 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 CFU mL-1 of E. coli in water within 6 hours by targeting the bacteria's 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.

Flow-through colorimetric assay for detection of nucleic acids in plasma.

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.

Colorimetric Detection of Salivary α-Amylase Using Maltose as a Noncompetitive Inhibitor for Polysaccharide Cleavage.

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.

Visual detection of Al(3+) ions using conjugated copolymer-ATP supramolecular complex.

A colorimetric Al(3+) 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 Al(3+) in deionized (DI) and tap water. The electrostatic disassembly of CP-ATP upon addition of Al(3+) led to an evident visual color change. The lowest concentration of Al(3+) 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 Al(3+) in tap water. The proposed methodology showed selective and sensitive detection for Al(3+) in analytically relevant concentration ranges without involving sophisticated instrumentation, illustrating the applicability for on-site drinking water monitoring.

Immunosensor based on carbon nanotube/manganese dioxide electrochemical tags.

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.

Naked eye detection of lung cancer associated miRNA by paper based biosensing platform.

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.