Search

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.

Standard detection methods for nucleic acids, an important class of diagnostic biomarkers, are often laborious and cumbersome. In need for development of facile methodologies, localized surface plasmon resonance (LSPR) assays have been widely explored for both spectroscopic and visual detection of nucleic acids. Our sensing approach is based on monitoring changes in the LSPR band due to interaction between peptide nucleic acid (PNA) and plasmonic nanoparticles (NPs) in the presence/absence of target nucleic acid. We have investigated the importance of tuning the stoichiometry of PNA to NPs to enable "naked-eye" detection of nucleic acids at clinically relevant concentration ranges. Assaying in plasma is achieved by incorporation of silver in gold NPs (AuNPs) via an alloying process. The synthesized gold/silver alloy NPs reduce nonspecific adsorption of proteinaceous interferents in plasma. Furthermore, the gold/silver alloy NPs absorb in the most sensitive cyan to green transition zone (∼500 nm) yielding highly competitive visual limits of detection (LODs). The visual LOD (calculated objectively using the ΔE algorithm) for a model microRNA (mir21) using a productive combination of stoichiometric tuning of the PNA to NP ratio and compositional tuning of the NPs in buffer and plasma extract equals 200 pM (∼250 times lower than existing reports) and 3 nM, respectively. We envision that the proposed LSPR assay based on Au0.8Ag0.2NPs offers an avenue for rapid and sensitive on-site detection of nucleic acids in complex matrixes in combination with efficient target extraction kits.

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. Ministry of Education (MOE) The authors wish to acknowledge funding support from Tier 1, MOE -RG 82/12, and NITHM Exploratory Research grant M4081989.070.

Urinalysis enables non-invasive point-of-care (POC) testing of numerous biomarkers at their physiological and elevated levels, obviating the need for sophisticated equipment or trained personnel. POC urinalysis is used to identify biomarkers that are rich in urine (greater than 1 μM), such as lactate, uric acid, glucose, ions, and adenosine. Urine also contains biomarkers such as small molecules, nucleic acids, neurotransmitters, and drugs in trace amounts (less than 1 μM). These biomarkers are of significant importance for health care monitoring, diagnosis of various disorders (cancer, metabolic diseases, etc.) and illicit drug control (cocaine, steroids, etc.). While POC detection of urinary biomarkers at higher concentration (μM to mM) levels is feasible, direct assaying of biomarkers in nM to fM levels is challenging, as assay responses are typically masked by interferences from the urine sample matrix. This report is a consolidated review of emerging trends and challenges in the POC urinalysis for detecting biomarkers that are less abundant in urine. The sensing mechanisms, analytical device fabrication, discrete and integrated sample pre-treatment procedures for POC assaying of urinary markers in trace amounts are elaborated. Subsequently, the utilization of smart data analytics for facilitating personalized urinalysis is presented. A comprehensive outlook on associated challenges in POC urinalysis of biomarkers in trace amounts is further provided, which would facilitate the advancement of POC urinalysis for a wide range of healthcare applications.

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

Standard detection methods for nucleic acids, an important class of diagnostic biomarkers, are often laborious and cumbersome. In need for development of facile methodologies, localized surface plasmon resonance (LSPR) assays have been widely explored for both spectroscopic and visual detection of nucleic acids. Our sensing approach is based on monitoring changes in the LSPR band due to interaction between peptide nucleic acid (PNA) and plasmonic nanoparticles (NPs) in the presence/absence of target nucleic acid. We have investigated the importance of tuning the stoichiometry of PNA to NPs to enable "naked-eye" detection of nucleic acids at clinically relevant concentration ranges. Assaying in plasma is achieved by incorporation of silver in gold NPs (AuNPs) via an alloying process. The synthesized gold/silver alloy NPs reduce nonspecific adsorption of proteinaceous interferents in plasma. Furthermore, the gold/silver alloy NPs absorb in the most sensitive cyan to green transition zone (∼500 nm) yielding highly competitive visual limits of detection (LODs). The visual LOD (calculated objectively using the Δ

Over the past few decades, colorimetric assays have been developed for cost-effective and rapid on-site urinalysis. Most of these assays were employed for detection of biomarkers such as glucose, uric acid, ions, and albumin that are abundant in urine at micromolar to millimolar levels. In contrast, direct assaying of urinary biomarkers such as glycated proteins, low-molecular-weight reactive oxygen species, and nucleic acids that are present at significantly lower levels (nanomolar to picomolar) remain challenging due to the interferences from the urine sample matrix. State-of-the-art assays for detection of trace amounts of urinary biomarkers typically utilize time-consuming and equipment-dependent sample pretreatment or clean-up protocols prior to assaying, which limits their applicability for on-site analysis. Herein, we report a colorimetric assay for on-site detection of trace amount of generic biomarkers in urine without involving tedious sample pretreatment protocols. The detection strategy is based on monitoring the changes in optical properties of poly(3-(4-methyl-3'-thienyloxy)propyltriethylammonium bromide) upon interacting with an aptamer or a peptide nucleic acid in the presence and absence of target biomarkers of relevance for the diagnosis of metabolic complications and diabetes. As a proof of concept, this study demonstrates facile assaying of advanced glycation end products, 8-hydroxy-2'-deoxyguanosine and hepatitis B virus DNA in urine samples at clinically relevant concentrations, with limits of detection of ∼850 pM, ∼650 pM, and ∼ 1 nM, respectively. These analytes represent three distinct classes of biomarkers: (i) glycated proteins, (ii) low-molecular-weight reactive oxygen species, and (iii) nucleic acids. Hence, the proposed methodology is applicable for rapid detection of generic biomarkers in urine, without involving sophisticated equipment and skilled personnel, thereby enabling on-site urinalysis. At the end of the contribution, we discuss the opportunity to translate the homogeneous assay into a paper-based format. Ministry of Education (MOE) Nanyang Technological University The authors wish to acknowledge funding support from Tier 1, MOE -RG 82/12, and NITHM Exploratory Research grant M4081989.070. B.O.B. is supported by an Ong Tiong Tat Chair Professorship and MOE Tier 1 project 2017-T1-001- 139.

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

Conjugated polyelectrolytes (CPEs) have been widely used as reporters in colorimetric assays targeting nucleic acids. CPEs provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. Herein, we report a pixelated analysis approach for correlating colorimetric responses of CPE with nucleic acid concentrations down to 1 nM, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. The detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic CPE duplexes and double stranded nucleic acid-CPE triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. Cationic poly[N,N,N-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the CPE reporter deposited on a polyvinylidene fluoride (PVDF) membrane for nucleic acid assay. A smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of CPE on the PVDF membrane, followed by an analysis using the algorithm. The proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, RGB analysis. The obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation., Graphical abstract Image 1, Highlights • A cost effective sensory cartridge and an algorithm compatible with smart phone are developed. • Conjugated polyelectrolyte impregnated paper for detection of nucleic acid. • A pixelated assay to visualize nucleic acid down to concentration of 1 nM.

Liedberg, Bo/0000-0003-2883-6953, WOS: 000427204100023, PubMed: 29430928, A luminescent paper-based device for the visual detection of oxidative stress biomarkers is reported. The device consists of a polyvinylidene fluoride membrane impregnated with poly(3-alkoxy-4-methylthiophene) (PT) for colorimetric detection. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker associated with oxidative stress, is used as a model system for validating the proposed methodology. The detection strategy is based on monitoring the changes in optical properties of PT associated with its conformational changes upon interaction with an aptamer in the presence and in the absence of 8-OHdG. Fluorometric and colorimetric monitoring revealed linear responses for 8-OHdG concentrations between 50 pM and 500 nM (similar to 14 pg/mL to 140 ng/mL), with limits of detection of 300 pM and 350 pM, respectively for (n = 3). Colorimetric responses in artificial urine ascertained rapid, sensitive, and selective detection of 8-OHdG at clinically relevant (pM to nM) concentration levels. Furthermore, the proposed methodology enables point-of-care diagnostics for oxidative stress without requiring sophisticated instrumentation.

Here we report on the design and synthesis of cationic water-soluble thiophene copolymers as reporters for colorimetric detection of microRNA (miRNA) in human plasma. Poly(3-alkoxythiophene) (PT) polyelectrolytes with controlled ratios of pendant groups such as triethylamine/1-methyl imidazole were synthesized for optimizing interaction with target miRNA sequence (Tseq). Incorporation of specific peptide nucleic acid (PNA) sequences with the cationic polythiophenes yielded distinguishable responses upon formation of fluorescent PT-PNA-Tseq triplex and weakly fluorescent PT-Tseq duplex, thereby enabling selective detection of target miRNA. Unlike homopolymers of PT (hPT), experimental results indicate the possibility of utilizing copolymers of PT (cPT) with appropriate ratios of pendant groups for miRNA assay in complex matrices such as plasma. As an illustration, colorimetric responses were obtained for lung cancer associated miRNA sequence (mir21) in human plasma, with a detection limit of 10 nM, illustrating the feasibility of proposed methodology for clinical applications without involving sophisticated instrumentation. The described methodology therefore possesses high potential for low-cost nucleic acid assays in resource-limited settings.

A novel approach for miRNA assay using a cationic polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrobromide] (PT), immobilized on a quartz resonator is proposed. The cationic PT enables capturing of all RNA sequences in the sample matrix via electrostatic interactions, resulting in the formation of PT-RNA duplex structures on quartz resonators. Biotinylated peptide nucleic acid (b-PNA) sequences are subsequently utilized for the RNA assay, upon monitoring the PT-RNA-b-PNA triplex formation. Signal amplification is achieved by anchoring avidin coated nanoparticles to b-PNA in order to yield responses at clinically relevant concentration regimes. Unlike conventional nucleic acid assay methodologies that usually quantify a specific sequence of RNA, the proposed approach enables the assay of any RNA sequence in the sample matrix upon hybridization with a PNA sequence complementary to the RNA of interest. As an illustration, successful detection of mir21, (a miRNA sequence associated with lung cancer) is demonstrated with a limit of detection of 400 pM. Furthermore, precise quantification of mir21 in plasma samples is demonstrated without requiring PCR and sophisticated instrumentation.