Manoj Kumar Kanakasabapathy

Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection

Rapid, sensitive, and selective pathogen detection is of paramount importance in infectious disease diagnosis and treatment monitoring. Currently available diagnostic assays based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are time-consuming, complex, and relatively expensive, thus limiting their utility in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been used extensively in the development of rapid and sensitive diagnostic assays for pathogen detection and nucleic acid analysis and hold great promise for revolutionizing point-of-care molecular diagnostics. Here, we review novel LAMP-based lab-on-a-chip (LOC) diagnostic assays developed for pathogen detection over the past several years. We review various LOC platforms based on their design strategies for pathogen detection and discuss LAMP-based platforms still in development and already in the commercial pipeline. This review is intended as a guide to the use of LAMP techniques in LOC platforms for molecular diagnostics and genomic amplifications.

An automated smartphone-based diagnostic assay for point-of-care semen analysis

This work demonstrates that a low-cost smartphone accessory can be used for home-based male infertility screening. Sperm samples phoning in Although male infertility is as common as female infertility, it often goes undiagnosed because of socioeconomic factors such as stigma, high cost of testing, and availability of laboratory facilities. To facilitate the necessary testing, Kanakasabapathy et al. have designed a smartphone-based assay that can be performed at home or in a remote clinic without access to laboratory equipment. The assay uses an inexpensive device that attaches directly to a phone and is operated through a smartphone application. The accuracy of this approach was very similar to that of computer-assisted laboratory analysis, even when it was performed by untrained users with no clinical background, demonstrating its potential for use at home and in low-resource settings. Male infertility affects up to 12% of the world’s male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones.

Printed Flexible Plastic Microchip for Viral Load Measurement through Quantitative Detection of Viruses in Plasma and Saliva

We report a biosensing platform for viral load measurement through electrical sensing of viruses on a flexible plastic microchip with printed electrodes. Point-of-care (POC) viral load measurement is of paramount importance with significant impact on a broad range of applications, including infectious disease diagnostics and treatment monitoring specifically in resource-constrained settings. Here, we present a broadly applicable and inexpensive biosensing technology for accurate quantification of bioagents, including viruses in biological samples, such as plasma and artificial saliva, at clinically relevant concentrations. Our microchip fabrication is simple and mass-producible as we print microelectrodes on flexible plastic substrates using conductive inks. We evaluated the microchip technology by detecting and quantifying multiple Human Immunodeficiency Virus (HIV) subtypes (A, B, C, D, E, G, and panel), Epstein-Barr Virus (EBV), and Kaposi’s Sarcoma-associated Herpes Virus (KSHV) in a fingerprick volume (50 µL) of PBS, plasma, and artificial saliva samples for a broad range of virus concentrations between 102 copies/mL and 107 copies/mL. We have also evaluated the microchip platform with discarded, de-identified HIV-infected patient samples by comparing our microchip viral load measurement results with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) as the gold standard method using Bland-Altman Analysis.

Label-free electrical sensing of bacteria in eye wash samples: A step towards point-of-care detection of pathogens in patients with infectious keratitis.

The diagnosis of keratitis is based on visual exam, tissue cytology, and standard microbial culturing to determine the type of the infectious pathogen. To prescribe appropriate therapy, it is important to distinguish between bacterial, fungal, and viral keratitis, as the treatments are quite different. Diagnosis of the causative organism has a substantial prognostic importance. Further, timely knowledge of the nature of the pathogen is also critical to adapt therapy in patients unresponsive to empiric treatment options, which occurs in 10% of all cases. Currently, the identification of the nature of the pathogen that causes keratitis is achieved via microbial culture screening, which is laboratory-based, expensive, and time-consuming. The most frequent pathogens that cause the corneal ulcers are P. aeruginosa and S. aureus. Here, we report a microchip for rapid (<1h) detection of P. aeruginosa (6294), S. aureus(LAC), through on-chip electrical sensing of bacterial lysate. We evaluated the microchip with spiked samples of PBS with bacteria concentration between 101 to 108 CFU/mL. The least diluted bacteria concentration in bacteria-spiked samples with statistically significant impedance change was 10 CFU/mL. We further validated our assay by comparing our microchip results with the standard culture-based methods using eye washes obtained from 13 infected mice.

Rapid Real-Time Antimicrobial Susceptibility Testing with Electrical Sensing on Plastic Microchips with Printed Electrodes

Rapid antimicrobial susceptibility testing is important for efficient and timely therapeutic decision making. Due to globally spread bacterial resistance, the efficacy of antibiotics is increasingly being impeded. Conventional antibiotic tests rely on bacterial culture, which is time-consuming and can lead to potentially inappropriate antibiotic prescription and up-front broad range of antibiotic use. There is an urgent need to develop point-of-care platform technologies to rapidly detect pathogens, identify the right antibiotics, and monitor mutations to help adjust therapy. Here, we report a biosensor for rapid (<90 min), real time, and label-free bacteria isolation from whole blood and antibiotic susceptibility testing. Target bacteria are captured on flexible plastic-based microchips with printed electrodes using antibodies (30 min), and its electrical response is monitored in the presence and absence of antibiotics over an hour of incubation time. We evaluated the microchip with Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) as clinical models with ampicillin, ciprofloxacin, erythromycin, daptomycin, gentamicin, and methicillin antibiotics. The results are compared with the current standard methods, i.e. bacteria viability and conventional antibiogram assays. The technology presented here has the potential to provide precise and rapid bacteria screening and guidance in clinical therapies by identifying the correct antibiotics for pathogens.

Electrical response of a B lymphoma cell line latently infected with Kaposi's sarcoma herpesvirus

Certain viruses, such as herpesviruses, are capable of persistent and latent infection of host cells. Distinguishing and separating live, latently infected cells from uninfected cells is not easily attainable using current approaches. The ability to perform such separation would greatly enhance the ability to study primary, infected cells and potentially enable elimination of latently infected cells from the host. Here, the dielectrophoretic response of B cells infected with Kaposis sarcoma-associated herpesvirus (KSHV) were investigated and compared to uninfected B cells. We evaluated the effect of applied voltage, signal frequency, and flow rate of the sample on the cell capture efficiency. We achieved 37.1% ± 8.5% difference in capture efficiencies between latently KSHV-infected and uninfected BJAB B lymphoma cells at the chip operational conditions of 1V, 50 kHz and 0.02 μl/min sample flow rate. Our results show that latently infected B lymphoma cells demonstrated significantly different electrical response compared to uninfected B cells and DEP-based microchips can be potentially used for sorting latently infected cells based on their electrical properties.

Hybrid Paper–Plastic Microchip for Flexible and High‐Performance Point‐of‐Care Diagnostics

A low-cost and easy-to-fabricate microchip remains a key challenge for the development of true point-of-care (POC) diagnostics. Cellulose paper and plastic are thin, light, flexible, and abundant raw materials, which make them excellent substrates for mass production of POC devices. Herein, a hybrid paper-plastic microchip (PPMC) is developed, which can be used for both single and multiplexed detection of different targets, providing flexibility in the design and fabrication of the microchip. The developed PPMC with printed electronics is evaluated for sensitive and reliable detection of a broad range of targets, such as liver and colon cancer protein biomarkers, intact Zika virus, and human papillomavirus nucleic acid amplicons. The presented approach allows a highly specific detection of the tested targets with detection limits as low as 102 ng mL-1 for protein biomarkers, 103 particle per milliliter for virus particles, and 102 copies per microliter for a target nucleic acid. This approach can potentially be considered for the development of inexpensive and stable POC microchip diagnostics and is suitable for the detection of a wide range of microbial infections and cancer biomarkers.

Motion-Based Immunological Detection of Zika Virus Using Pt-Nanomotors and a Cellphone

Zika virus (ZIKV) infection is an emerging pandemic threat to humans that can be fatal in newborns. Advances in digital health systems and nanoparticles can facilitate the development of sensitive and portable detection technologies for timely management of emerging viral infections. Here we report a nanomotor-based bead-motion cellphone (NBC) system for the immunological detection of ZIKV. The presence of virus in a testing sample results in the accumulation of platinum (Pt)-nanomotors on the surface of beads, causing their motion in H2O2 solution. Then the virus concentration is detected in correlation with the change in beads motion. The developed NBC system was capable of detecting ZIKV in samples with virus concentrations as low as 1 particle/μL. The NBC system allowed a highly specific detection of ZIKV in the presence of the closely related dengue virus and other neurotropic viruses, such as herpes simplex virus type 1 and human cytomegalovirus. The NBC platform technology has the potential to be used in the development of point-of-care diagnostics for pathogen detection and disease management in developed and developing countries.

DNA engineered micromotors powered by metal nanoparticles for motion based cellphone diagnostics

HIV-1 infection is a major health threat in both developed and developing countries. The integration of mobile health approaches and bioengineered catalytic motors can allow the development of sensitive and portable technologies for HIV-1 management. Here, we report a platform that integrates cellphone-based optical sensing, loop-mediated isothermal DNA amplification and micromotor motion for molecular detection of HIV-1. The presence of HIV-1 RNA in a sample results in the formation of large-sized amplicons that reduce the motion of motors. The change in the motors motion can be accurately measured using a cellphone system as the biomarker for target nucleic acid detection. The presented platform allows the qualitative detection of HIV-1 (n = 54) with 99.1% specificity and 94.6% sensitivity at a clinically relevant threshold value of 1000 virus particles/ml. The cellphone system has the potential to enable the development of rapid and low-cost diagnostics for viruses and other infectious diseases.Micromotors have a range of potential healthcare applications. Here, the authors describe the development of a metal nanoparticle DNA micromotor which can be used to detect human HIV-1 by a change in the motion of the micromotors, monitored by cell phone camera, triggered by binding of HIV-1 RNA.