Point of Care Diagnostics

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.

Rapid point of care tests for bacterial infection diagnosis are of great importance to reduce the misuse of antibiotics and burden of antimicrobial resistance. Here, we have successfully combined a new class of non-biological binder molecules with electrochemical impedance spectroscopy (EIS)-based sensor detection for direct, label-free detection of Gram-positive bacteria making use of the specific coil-to-globule conformation change of the vancomycin-modified highly branched polymers immobilized on the surface of gold screen-printed electrodes upon binding to Gram-positive bacteria. Staphylococcus carnosus was detected after just 20 min incubation of the sample solution with the polymer-functionalized electrodes. The polymer conformation change was quantified with two simple 1 min EIS tests before and after incubation with the sample. Tests revealed a concentration dependent signal change within an OD600 range of Staphylococcus carnosus from 0.002 to 0.1 and a clear discrimination ...

The current COVID-19 pandemic outbreak poses a serious threat to public health, demonstrating the critical need for the development of effective and reproducible detection tests. Since the RT-qPCR primers are highly specific and can only be designed based on the known sequence, mutation sensitivity is its limitation. Moreover, the mutations in the severe acute respiratory syndrome β-coronavirus (SARS-CoV-2) genome led to new highly transmissible variants such as Delta and Omicron variants. In the case of mutation, RT-qPCR primers cannot recognize and attach to the target sequence. This research presents an accurate dual-platform DNA biosensor based on the colorimetric assay of gold nanoparticles and the surface-enhanced Raman scattering (SERS) technique. It simultaneously targets four different regions of the viral genome for detection of SARS-CoV-2 and its new variants prior to any sequencing. Hence, in the case of mutation in one of the target sequences, the other three probes cou...

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

The mechanisms underlying the complications of mild traumatic brain injury, including post-concussion syndrome, post-impact catastrophic death, and delayed neurodegeneration remain poorly understood. This limited pathophysiological understanding has hindered the development of diagnostic and prognostic biomarkers and has prevented the advancement of treatments for the sequelae of mild traumatic brain injury. We aimed to characterize the early electrophysiological and neurovascular alterations following repetitive mild traumatic brain injury and sought to identify new targets for the diagnosis and treatment of individuals at risk of severe post-impact complications. We combined behavioural, electrophysiological, molecular, and neuroimaging techniques in a rodent model of repetitive mild traumatic brain injury. In humans, we used dynamic contrast-enhanced MRI to quantify blood–brain barrier dysfunction after exposure to sport-related concussive mild traumatic brain injury. Rats could ...

Textile and their composite-based functional sensors are extensively acknowledged and preferred detection platforms in recent times. Developing suitable methodologies for fabricating textile sensors can be achieved either by integration of conductive fibers and yarns into textiles using technologies such as weaving, knitting and embroidery; or by functionalization of textile materials with conductive nanomaterials/inks using printing or coating methods. Textile materials are gaining enormous attention for fabricating soft lab-on-fabric devices due to their unique features such as high flexibility, wear and wash resistance, mechanical strength and promising sensing performances. Owing to these collective properties, textile-based electrochemical transducers are now showcasing rapid and accurate electrical measurements towards real time point-of-care diagnostics and environmental monitoring applications. The present review provides a brief overview of key progress made in the field of developing textile materials and their composites-based electrochemical sensors and biosensors in recent years where electrode configurations are specifically based on either natural or synthetic fabrics. Different ways to fabricate and functionalize textiles for their application in electrochemical analysis are briefly discussed. The review ends with a conclusive note focusing on the current challenges in the fabrication of textile-based stable electrochemical sensors and biosensors.

We present a microfluidic device for selective separation and extraction of molecules based on their diffusivity. The separation relies on electroosmotically-driven bidirectional flows in which high diffusivity species experience a net-zero velocity, and lower diffusivity species are advected to a collection reservoir. The device can operate continuously and is suitable for processing low sample volumes. Using several model systems, we showed that the extraction efficiency of the system is maintained at more than 90% over tens of minutes, with a purity of more than 99%. We demonstrate the applicability of the device to the extraction of genomic DNA from short DNA fragments.

Background: Coronavirus disease 2019 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is highly transmissible. Early and rapid testing is necessary to effectively prevent and control the outbreak. Detection of SARS-CoV-2 antibodies with lateral flow immunoassay can achieve this goal. Antibody detection is especially effective for the detection of asymptomatic infection.Methods: In this study, SARS-CoV-2 nucleoprotein was expressed by E. coli and purified by affinity chromatography. We used the highly stable and sensitive selenium nanoparticle as the labeling probe coupled with the SARS-CoV-2 nucleoprotein to prepare a new SARS-CoV-2 antibody (IgM and IgG) detection kit. The sensitivity and specificity of the kit were verified by plasma of COVID-19 patients and health persons. Separate detection of IgM and IgG, such as in this assay, was performed in order to reduce mutual interference and improve the accuracy of the test...

The coronavirus disease 2019 (COVID-19) pandemic has taught us much about our weaknesses in the management of infectious disease outbreaks. A key lesson has been the need for more effective point-of-care diagnostic tools that not only produce rapid and reliable results, but also facilitate decentralised testing to avoid overwhelming central test facilities when demand peaks in an outbreak. Microneedle devices can be inserted painlessly into the skin to detect biomolecules in the epidermal and dermal layers. They have been used to detect biomarkers both in the interstitial fluid and the capillary blood. Importantly, they are amenable to self-administration. In this article, we provide an overview of existing microneedle-based diagnostic technologies and discuss how they may be built upon to provide effective diagnostic tools for infectious diseases in general.

A lateral flow based detection method for ascertaining the presence of soymilk in whole bovine milk has been described. The method uses commercially available rabbit anti-soy protein antibodies conjugated to gold nanoparticles (AuNPs) wherein soymilk protein in adulterated milk and soymilk protein at test line competes for limited antibodies. At control line, anti-rabbit immunoglobulin was immobilized for ensuring flow properties of antibody-conjugated AuNPs. Absence or diminished intensity of band at test line indicates presence of soymilk in milk. The soymilk detection limit was 1.75% (v/v) in whole bovine milk and results are available in 5 min. Constructed lateral flow device can be used for on-spot examination of soymilk in milk.

Recently the field of paper-based microfluidics has garnered significant interest due to the desire to develop very low cost diagnostic devices [1]. Critical to the development of the field is the availability of tools to monitor, or measure, liquid flow in paper, a porous and opaque material. Despite recent efforts to monitor flow in paper, there is yet a technique that is both easy to use and provides detailed flow information in real-time. To address this critical need, we demonstrate a real-time, non-optical technique, based on electrochemical velocimetry, to monitor liquid flow in paper samples.

Despite the transfer of COVID-19 from the pandemic to control, we are still in a state of uncertainty about long-term success. Therefore, there is a great need for rapid and sensitive diagnostics to sustain the control status. After several optimization trials, we developed lateral flow test (LFT) strips for rapid detection of SARS-CoV-2 spike 1 (S1) antigen in saliva samples. For signal enhancement of our developed strips, we applied dual gold conjugates. Gold-labeled anti-S1 nanobodies (Nbs) were employed as S1 detector conjugate, while gold-labeled angiotensin-converting enzyme 2 (ACE2) was used as S1 capturing conjugate. In a parallel strip design, we used an anti-S1 monoclonal antibody (mAb) as an antigen detector instead of anti-S1 Nbs. Saliva samples were collected from 320 symptomatic subjects (180 RT-PCR confirmed positive cases and 140 confirmed negative cases) and were tested with the developed strips. In early detection for positive samples with cycle threshold (Ct ≤ 30)...

Dengue virus, a family member, has emerged as a major worldwide health concern, making its early diagnosis imperative. Lateral flow immunoassays have been widely employed for point-of-care diagnosis of dengue because of their rapid naked eye readouts, ease of use, and cost-effectiveness. However, they entail a drawback of low sensitivity, limiting their usage in clinical applications. Herein, we report a novel lateral flow immunoassay for detection of dengue leveraging on the benefits of gold decorated graphene oxide sheets as detection labels and a tapered nitrocellulose membrane. The developed assay allows for rapid (10 min) and sensitive detection of dengue NS1 with a detection limit of 4.9 ng mL, ∼11-fold improvement over the previously reported values. Additionally, the clinical application of the developed assay has been demonstrated by testing it for dengue virus spiked in human serum. The reported lateral flow immunoassay shows significant promise for early and rapid detecti...

From acute to chronic hepatitis, cirrhosis, and hepatocellular cancer, hepatitis B infection causes a broad spectrum of liver diseases. Molecular and serological tests have been used to diagnose hepatitis B-related illnesses. Due to technology limitations, it is challenging to identify hepatitis B infection cases at an early stage, particularly in a low- and middle-income country with constrained resources. Generally, the gold-standard methods to detect hepatitis B virus (HBV) infection requires dedicated personnel, bulky, expensive equipment and reagents, and long processing times which delay the diagnosis of HBV. Thus, lateral flow assay (LFA), which is inexpensive, straightforward, portable, and operates reliably, has dominated point-of-care diagnostics. LFA consists of four parts: a sample pad where samples are dropped; a conjugate pad where labeled tags and biomarker components are combined; a nitrocellulose membrane with test and control lines for target DNA-probe DNA hybridiz...

An integrated centrifugal microfluidic device was developed to preconcentrate and detect hazardous mercury (II) in water with ionic liquid as environmentally friendly extractant. An automatically salt-controlled ionic liquid dispersive liquid–liquid microextraction on a centrifugal microfluidic device was designed, fabricated, and characterized. The entire liquid transport mixing and separation process was controlled by rotation speed, siphon valves, and capillary valves. Still frame images on the rotating device showed the process in detail, revealing the sequential steps of mixing, siphon priming, transportation between chambers, and phase separation. The preconcentration of red dye could be clearly observed with the naked eye. By combining fluorescence probe and microscopy techniques, the device was tested to determine ppb-level mercury (II) in water, and was found to exhibit good linearity and low detection limit.

Laser micromachining has emerged as a promising technique for mass production of microfluidic devices. However, control and optimization of process parameters, and design of substrate materials are still ongoing challenges for the widespread application of laser micromachining. This article reports a systematic study on the effect of laser system parameters and thermo-physical properties of substrate materials on laser micromachining. Three dimensional transient heat conduction equation with a Gaussian laser heat source was solved using finite element based Multiphysics software COMSOL 5.2a. Large heat convection coefficients were used to consider the rapid phase transition of the material during the laser treatment. The depth of the laser cut was measured by removing material at a pre-set temperature. The grid independent analysis was performed for ensuring the accuracy of the model. The results show that laser power and scanning speed have a strong effect on the channel depth, whi...