Point of care diagnostics for carbonyl/oxidative stress biomarkers

Multiple triggers like exogenous and endogenous factors could affect the equilibrium levels of free radicals and reactive oxygen species in humans. For instance, elevated concentration levels of reactive carbonyl species (carbonyl stress) or reactive oxygen species (oxidative stress) damages a myriad of important biological molecules such as proteins, lipids, carbohydrates and nucleic acids. Damage to these molecules causes cell death and tissue injury, thus contributing to various disorders like type 2 diabetes mellitus (T2D), cancer, and cardiovascular diseases. Therefore, assays for stress related biomarkers could assist in the early diagnosis and could be predictive for disease prognosis. The conventional assay methodologies such as chromatographic and electrophoretic techniques are cumbersome, skill intensive and require expensive instrumentation. Thus, there is sufficient scope to develop a point of care (POC) assay for these biomarkers such that advantages of low cost, fast response time and high sensitivity as well as selectivity can be realized. Carbonyl/Oxidative stress plays a major role in the onset and progression of T2D, and a simple, low cost way of measurement of carbonyl/oxidative stress biomarkers can be beneficial in screening patients for early diagnosis of T2D and its complications thereby assisting current clinical practice. The focus of this thesis was to develop colorimetric assays for monitoring carbonyl/oxidative stress biomarkers associated with T2D. Cationic polythiophene (PT); a water-soluble conjugated polymer was used as a luminescent reporter for the optical detection of these biomarkers. The developed assay consists of two modules; a sample fractioning unit; a filter paper (FUSION 5) for removal of large molecules from clinical samples without requiring external stimuli, and a transduction unit utilizing polyvinylidene fluoride (PVDF) membrane impregnated with (PT) for colorimetric sensing in a flow-through format. The sensing strategy is based on monitoring the changes in optical properties of PT with its associated conformational changes when interacting with a peptide nucleic acid (PNA)/aptamer in the presence and in the absence of target biomarkers. As a proof of concept, the developed assay methodology utilizing PT and PNA/aptamer was validated using different carbonyl/oxidative stress biomarkers such as microRNA (mir21), DNA damage molecule; 8-hydroxy-2′-deoxyguanosine (8-OHdG) and Glyceraldehyde derived Advanced Glycation End Products (Gly-AGEs) without requiring tedious sample pre-treatment and clean up protocols. Colorimetric responses for these biomarkers were obtained at clinically relevant concentrations and could therefore find applications in relation to the management of metabolic diseases such as T2D in a clinical setting as well as in the patient’s home, thus providing attractive and affordable healthcare solutions for field diagnostics.Doctor of Philosoph

Passive blood plasma separation using microfluidics

Blood is an important element in the human body. Blood test is the most commonly performed medical test as it provides the information regarding the health of a person. Blood Plasma separation is very essential; otherwise it can cause testing errors. The conventional laboratorial test requires expensive equipment and this set up is not well equipped in developing countries. The laboratorial tests are also time consuming and require large volume of blood. So it is essential to have a miniaturized device for low costs and less time consuming blood test.​Master of Science (Biomedical Engineering

Luminescent device for the detection of oxidative stress biomarkers in artificial urine

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 (∼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.Tier 1 MOE-RG (82/12); NITHM (M4081989.070.500000

Current trends and challenges in point-of-care urinalysis of biomarkers in trace amounts

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

Colorimetric urinalysis for on-site detection of metabolic biomarkers

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 UniversityThe 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

Visual detection of Al3+ ions using conjugated copolymer-ATP supramolecular complex

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, Singapor

Luminescent Device for the Detection of Oxidative Stress Biomarkers in Artificial Urine

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 (∼14 pg/mL to 140 ng/mL), with limits of detection of ∼300 pM and  ∼350 pM, respectively for (<i>n</i> = 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

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.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

Outer‐Membrane Protease (OmpT) Based E. coli

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 versionThis 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