Hybrid point-of-care devices for visual detection of biomarkers and drugs

Early diagnostics is a crucial part of clinical practice offering a rapid and convenient way to investigate and quantify the presence of key biomarkers related to specific pathologies and increasing the chance of successful treatments. In this regard, point-of-care testing (POCT) shows several advantages enabling simple and rapid analyses, allowing for real-time results, and permitting home testing. Metallic nanoparticles (NPs), like gold NPs (AuNPs), can be beneficially integrated into POC devices thanks to their tunable plasmonic properties which provide a naked-eye read-out. Moreover, the high sensitivity of NPs enables the detection of biomarkers in non-invasive fluids where the concentrations are typically low. These biofluids, like saliva and urine, are functionally equivalent to serum in reflecting the physiological state of the body, whilst they are easier to handle, collect, and store. In this thesis, I first reported the design and development of a colorimetric strategy based on the morphological change of multibranched plasmonic AuNPs, aimed at detecting glucose in saliva. The sensing approach relied on a target-induced reshaping process which involves the oxidation of the NP tips and the transformation into a spherical shape, characterized by a naked-eye detectable blue-to-pink color change. The platform proved to be beneficial in the early and non-invasive diagnosis of hyperglycemia. The successful technological transfer on a solid substrate paved the way for the realization of a dipstick prototype for home testing. Then, the strategy was adapted to other biomarkers, leading to the development of a multiplexing test for the simultaneous detection of three salivary analytes (cholesterol, glucose, and lactate). This multiplexing assay enabled to save reagents, costs, and time, whilst increasing the overall clinical value of the test. Exploiting the microfluidics applied on a paper sheet, I realized a monolithic and fully integrated POC device, through a low-cost and fast CO2 laser cutter. The platform showed excellent selectivity and multiplexing ability, with negligible interferences. The second part of my thesis was focused on the development of POC devices for the detection of anticancer drug contaminations in water solutions and urine samples. Antiblastic agents have revealed high toxicity for the exposed healthcare workers who prepare and administer these drugs in occupational environments. Hence, continuous monitoring is highly required, and POCT shows tremendous potential in this context. With this aim, I realized a lateral-flow (LF) device for the assessment of doxorubicin contamination, using the fluorescent properties of the drug for naked-eye detection. The pharmacological recognition of the DNA probe was exploited to overcome the lack of anti-doxorubicin antibodies. The highly sensitive strategy was successfully adapted to a real urine sample, without resorting to complex pretreatment procedures. Then, I developed a competitive LF device for the detection of methotrexate (MTX). AuNPs were employed as the label molecules and the pharmacological competition of folic acid and MTX for the capture enzyme was exploited as the recognition mechanism, instead of costly antibodies. Despite the sensitivity requires further improvements, the strategy showed fast and reliable results, demonstrating a high potential for workers’ safety control

Colorimetric paper-based sensors against cancer biomarkers

Cancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls.This research was funded by Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UIDB/04469/2020 unit and through Mariana Carneiro PhD grant reference SFRH/BD/131959/2017.info:eu-repo/semantics/publishedVersio

Colorimetric Paper-Based Sensors against Cancer Biomarkers

Cancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls.This research was funded by Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UIDB/04469/2020 unit and through Mariana Carneiro PhD grant reference SFRH/BD/131959/2017.info:eu-repo/semantics/publishedVersio

A novel colorimetric biosensor based on non-aggregated Au@Ag core–shell nanoparticles for methamphetamine and cocaine detection

We report a novel colorimetric biosensor based on non-aggregation Au@Ag core-shell nanoparticles to detect methamphetamine and cocaine. The biosensor consisted of a reporter probe (RP) that is a specific single-stranded DNA (ssDNA) sequence coated on Au@Ag nanoparticles, a capture probe (CP) conjugated with magnetic beads, and an illicit drug-binding DNA aptamer (Apt). Au@Ag nanoparticles were synthesized by seed growth and characterized by scanning electron microscope (SEM), high-resolution transmission electron microscopy (HR-TEM), and UV–vis spectra. Methamphetamine (METH) was used as an example to evaluate the feasibility of the biosensor and to optimize the detection conditions. We demonstrated that this sensing platform was able to detect as low as 0.1 nM (14.9 ng L−1) METH with a negligible interference from other common illicit drugs. Various concentrations of METH were spiked into urines, and the biosensor yielded recoveries more than 83.1%. In addition, the biosensor also showed a high sensitivity to detect cocaine. These results demonstrated that our colorimetric sensor holds promise to be implemented as a visual sensing platform to detect multiple illicit drugs in biological samples and environmental matrices

DEVELOPMENT OF PAPER-BASED ANALYTICAL DEVICE FOR DETECTING DIAZEPAM IN URINE

Objective: The aim of this study was to develop Paper-based Analytical Devices (PADs) with colorimetric method as a presumptive test for detecting diazepam in urine Methods: Colorimetric method was used as a principle of this study. PADs were fabricated with wax-printing methods. Chosen colorimetric reagent was tested for selectivity with hydromorphone and codeine; and sensitivity by measuring the absorbance with UV-Vis spectrophotometer. PADs were tested for its sensitivity and stability. The intensity of color developed on PADs are measured with ImageJ. The ability of PADs to detect diazepam in urine was simulated with testing spiked urine sample to the PADs Results: Zimmermann gave the most obvious prominent color change from colorless to purple-red color out of the four reagents. PAD is selective to diazepam when tested with hydromorphone and codeine. PAD is sensitive with a cut-off concentration at 100 ppm. PAD can detect diazepam in urine with the highest recovery percent at 92.8%±4,6 Conclusion: It can be concluded that PAD is quite selective and sensitive to detect diazepam in urine and can be done easily and fast for onsite analysi

Towards Stable Electrochemical Sensing for Wearable Wound Monitoring

Wearable biosensing has the tremendous advantage of providing point-of-care diagnosis and convenient therapy. In this research, methods to stabilize the electrochemical sensing response from detection of target biomolecules, Uric Acid (UA) and Xanthine, closely linked to wound healing, have been investigated. Different kinds of materials have been explored to address such detection from a wearable, healing platform. Electrochemical sensing modalities have been implemented in the detection of purine metabolites, UA and Xanthine, in the physiologically relevant ranges of the respective biomarkers. A correlation can be drawn between the concentrations of these bio-analytes and wound severity, thus offering probable quantitative insights on wound healing progression. These insights attempt to contribute in reducing some impacts of the financial structure on the healthcare economy associated with wound-care. An enzymatic electrochemical sensing system was designed to provide quick response at a cost-effective, miniaturized scale. Robust enzyme immobilization protocols have assisted in preserving enzyme activity to offer stable response under relevant variations of temperature and pH, from normal. Increased hydrophilicity of the sensor surface using corona plasma, has assisted in improving conductivity, thus allowing for increased electroactive functionalization and loading across the substrate’s surface. Superior sensor response was attained from higher loading of nanomaterials (MWCNT/AuNP) and enzymes (UOx/XO) employed in detection. Potentiometric analyses of the nanomaterial modified enzymatic biosensors were conducted using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) modalities. Under relevant physiological conditions, the biosensor was noted to offer a variation in response between 10 % and 30 % within a week. Stable, reproducible results were obtained from repeated use of the biosensor over multiple days, also offering promise for continuous monitoring. Shelf life of the biosensor was noted to be more than two days with response retained by about 80 % thereafter. Secondary analyses have been performed utilizing the enzymatic biosensor to explore the feasibility of target biomarker detection from clinical extracts of different biofluids for wound monitoring. Biosensor response evaluation from the extracts of human wound exudate, and those obtained from perilesional and healthy skin, provided an average recovery between 107 % and 110 % with a deviation within (+/-) 6 %. Gradual decrease in response (10-20 %) was noted in detection from extracts further away from injury site. Increased accumulation of biofluids on the sensor surface was studied to explore sensor response stability as a function of sample volume. With a broad linear range of detection (0.1 nM – 7.3 mM) and detection limits lower than the physiological concentrations, this study has assessed the viability of stable wound monitoring under physiologically relevant conditions on a wearable platform

Paper-Based Point-of-Care Tools for Blood Testing

Early detection of malignant disease is crucial for timely diagnosis and effective medical intervention, which significantly increases survival rates and reduce financial burden on patients. Biomarkers are becoming increasingly important in detection of malignant diseases, because they can be employed for indicating diseases, predicting risks and monitoring the progression of diseases. In addition, biomarkers show up at early stages of diseases in human tissues and fluids (e.g., blood, urine and saliva), which shows great promise for early disease detection. In this dissertation, paper-based lateral flow strips (PLFSs) have been developed for the detection of disease biomarkers, including protein biomarkers and microRNA (miRNA) biomarkers from clinical samples and whole blood samples. Among most of the reported biosensors, PLFSs appear to be an effective tool for providing access to point-of-care (POC) applications, due to low cost, fast response, portability and ease of use. In addition, paper is compatible with biological samples, which allows its application in analyzing various biomolecules. However, conventional PLFSs exhibit insufficient sensitivity and poor interference resistance. In particular whole blood samples, which contain numerous interference biomolecules, substantially affect detection accuracy and specificity. In this dissertation, several strategies have been employed to solve the problems, including introducing PLFSs with ultrasensitive techniques, meanwhile modifying PLFSs with functional nanomaterials and paper accessory unit to reduce the interference biomolecules from human fluids. In summary, five chapters will be demonstrated: (1) Surface-enhanced Raman scattering (SERS) technique modified PLFSs for protein biomarker detection in clinical blood plasma samples. In this chapter, silica coated SERS nanoparticles (NPs) have been developed for improving Raman signal and detection sensitivity, at the same time, silica coating of the SERS NPs substantially improve the stability of the NPs in complex human fluids. As a result, the developed SERS-PLFS can realize direct detection of neuron-specific enolase (NSE) from clinical blood plasma samples of traumatic brain injury (TBI) patients. The test results of the SERS-PLFSs were compatible with those from the standard enzyme-linked immunospecific assay (ELISA) method; (2) Blood plasma separation unit (PSU) integrated PLFS for cancer protein biomarker detection from whole human blood sample. In this chapter, a paper based PSU was fabricated to efficiently retain red blood cells (RBCs) inside the unit to block their migration and meanwhile push the target protein contained plasma to the detection area of the PLFS. As a result, cancer protein biomarker-carcinoembryonic antigen (CEA) was successfully detected by the PSU-PLFS from whole blood samples; (3) Plasmonic chip and PSU integrated PLFS for protein biomarker detection from whole blood. In order to meet the high sensitivity demand, a gold nanopyramid array functionalized chip was integrated into a PLFS for amplifying Raman signal and ultrasensitive detection of TBI protein biomarker s-100β; while the PSU was utilized to reduce the RBCs interference from whole blood. As a result, compared with the result of the SERS-PLFS in Chapter 2, an improvement in LOD with two-order of magnitude was obtained; (4) Near-infrared fluorophores (NIRFs) functionalized PLFS for miRNA detection from blood plasma. In this chapter, NIRFs encapsulated silica nanoparticles were synthesized and incorporated into a PLFS for stroke biomarker miRNA-34 detection. Compared with a single fluorescent dye, the synthesized NIRF NPs encapsulated numerous fluorophores into one single silica nanoparticle, exhibiting amplified luminescent intensity. Moreover, the NIRF nanoparticles minimized the fluorescent background from biological matrices and test strip materials, which elevates signal-to-noise ratio and anti-interference capacity; (5) Duplex specific nuclease (DSN) based signal amplification strategy modified PLFS for ultrasensitive miRNA detection in blood plasma. In order to meet the high sensitivity demand of miRNA-34 measurement from the clinical blood plasma of stroke patient, the developed NIRFs-PLFS in Part 4 was further elevated with DSN modification for amplifying fluorescent signal. As a result, the DSN-PLFS exhibited an improvement in detection sensitivity with two-orders of magnitude in blood plasma

Advances in testing for sample manipulation in clinical and forensic toxicology - Part A: urine samples

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine

Colorimetric nanodiagnostics for Point-Of-Care applications: detection of salivary biomarkers and environmental contaminants

Nanomaterials offer many unique opportunities for the development of effective and rapid point-of-care (POC) devices to be exploited in many fields, including early diagnosis, health monitoring, and pollutant detection. In particular, gold nanoparticles (AuNPs) exhibit tunable catalytic and plasmonic properties, which are key enabling tools to design and develop innovative detection schemes in several sensing applications. The aim of this PhD project was the development of AuNPs-based colorimetric POCs to detect heavy metal ion contaminations and specific biomarkers in non-invasive biological fluids. First, we developed a novel strategy that exploits the combination of the plasmonic and catalytic properties of AuNPs to achieve an ultrafast (1 min) and sensitive colorimetric sensor for highly toxic methyl mercury. Taking advantage of the AuNP nanocatalyst to promote the rapid reduction of methyl mercury with nucleation on the particle surface and consequent aggregation-induced plasmonic shift, we were able to detect by naked-eye mercury contaminations as low as 20 ppb, which is relevant for food contaminations or biological fluid assessment. Moreover, an innovative and versatile platform, based on multibranched AuNPs, was developed for the detection of salivary biomarkers. Coupling etching and growing reactions in a reshaping process onto the nanostars surface, we created a customizable platform with boosted color change readout for fast detection of salivary glucose at low concentrations. The nanosensor performance was validated on samples from patients with diabetes, proving its potential as a novel non-invasive tool for frequent monitoring of glycaemia. As side project we also investigated the platinum nanoparticles enzymatic activity in a colorimetric sensor for inorganic mercury contamination monitoring in water sources

Community sewage sensors towards evaluation of drug use trends: detection of cocaine in wastewater with DNA-directed immobilization aptamer sensors

Illicit drug use has a global concern and effective monitoring and interventions are highly required to combat drug abuse. Wastewater-based epidemiology (WBE) is an innovative and cost-effective approach to evaluate community-wide drug use trends, compared to traditional population surveys. Here we report for the first time, a novel quantitative community sewage sensor (namely DNA-directed immobilization of aptamer sensors, DDIAS) for rapid and cost-effective estimation of cocaine use trends via WBE. Thiolated single-stranded DNA (ssDNA) probe was hybridized with aptamer ssDNA in solution, followed by co-immobilization with 6-mercapto-hexane onto the gold electrodes to control the surface density to effectively bind with cocaine. DDIAS was optimized to detect cocaine at as low as 10 nM with a dynamic range from 10 nM to 5 μM, which were further employed for the quantification of cocaine in wastewater samples collected from a wastewater treatment plant in seven consecutive days. The concentration pattern of the sampling week is comparable with that from mass spectrometry. Our results demonstrate that the developed DDIAS can be used as community sewage sensors for rapid and cost-effective evaluation of drug use trends, and potentially implemented as a powerful tool for on-site and real-time monitoring of wastewater by un-skilled personnel