Electrochemical sensors have the potential to achieve sensitive, specific, and low-cost detection of biomolecules--a capability that is ever more relevant to the diagnosis and monitored treatment of disease. The development of devices for clinical diagnostics based on electrochemical detection could provide a powerful solution for the routine use of biomarkers in patient treatment and monitoring and may overcome the many issues created by current methods, including the long sample-to-answer times, high cost, and limited prospects for lab-free use of traditional polymerase chain reaction, microarrays, and gene-sequencing technologies. In this Account, we summarize the advances in electrochemical biomolecular detection, focusing on a new and integrated platform that exploits the bottom-up fabrication of multiplexed electrochemical sensors composed of electrodeposited noble metals. We trace the evolution of these sensors from gold nanoelectrode ensembles to nanostructured microelectrodes (NMEs) and discuss the effects of surface morphology and size on assay performance. The development of a novel electrocatalytic assay based on Ru(3+) adsorption and Fe(3+) amplification at the electrode surface as a means to enable ultrasensitive analyte detection is discussed. Electrochemical measurements of changes in hybridization events at the electrode surface are performed using a simple potentiostat, which enables integration into a portable, cost-effective device. We summarize the strategies for proximal sample processing and detection in addition to those that enable high degrees of sensor multiplexing capable of measuring 100 different analytes on a single chip. By evaluating the cost and performance of various sensor substrates, we explore the development of practical lab-on-a-chip prototype devices. By functionalizing the NMEs with capture probes specific to nucleic acid, small molecule, and protein targets, we can successfully detect a wide variety of analytes at clinically relevant concentrations and speeds. Using this platform, we have achieved attomolar detection levels of nucleic acids with overall assay times as short as 2 min. We also describe the adaptation of the sensing platform to allow for the measurement of uncharged analytes--a challenge for reporter systems that rely on the charge of an analyte. Furthermore, the capabilities of this system have been applied to address the many current and important clinical challenges involving the detection of pathogenic species, including both bacterial and viral infections and cancer biomarkers. This novel electrochemical platform, which achieves large molecular-to-electrical amplification by means of its unique redox-cycling readout strategy combined with rapid and efficient analyte capture that is aided by nanostructured microelectrodes, achieves excellent specificity and sensitivity in clinical samples in which analytes are present at low concentrations in complex matrices.
There is increasing evidence that endoplasmic reticulum (ER) stress contributes to the development of atherosclerosis in diabetes mellitus. The purpose of this study was to determine the effects of increased hexosamine biosynthesis pathway (HBP) flux on ER stress levels and the complications of ER stress associated with diabetes and atherosclerosis in hepatic cells. Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, was overexpressed in HepG2 cells by use of an adenoviral expression system. The ER stress response and downstream effects, including activation of lipid and inflammatory pathways, were determined using real-time PCR, immunoblot analysis, and cell staining techniques. GFAT overexpression resulted in increased expression of ER stress markers, including Grp78, Grp94, calreticulin, and GADD153, relative to cells infected with an empty adenoviral vector. In addition, GFAT overexpression promoted lipid, but not cholesterol, accumulation in hepatic cells as well as inflammatory pathway activation. Treatment with 6-diazo-5-oxo-norleucine, a GFAT antagonist, blocked the effects of GFAT overexpression. Consistent with our in vitro data, hyperglycemic mice presented with elevated markers of hepatic ER stress, glucosamine and lipid accumulation. Together, these data suggest that HBP flux-induced ER stress plays a role in the development of hepatic steatosis and atherosclerosis under conditions of hyperglycemia.
Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in a number of complications associated with diabetes mellitus including micro‐ and macrovascular dysfunction. In this study we examine ER stress levels in blood cells isolated from human subjects with metabolic syndrome and in healthy controls. Total RNA and protein were isolated from leukocytes and the levels of specific ER stress markers were quantified by real‐time‐PCR and immunoblot analysis. Our results indicate that, compared to healthy controls, individuals with metabolic syndrome have elevated mRNA levels of genes indicative of ER stress; including spliced XBP‐1 (sXBP‐1), Grp78, and CHOP. Induced ER stress levels correlate with blood glucose but not plasma lipid concentration. Furthermore, in healthy individuals, a standard 75 g oral glucose challenge produced a significant elevation in spliced XBP‐1 (1.3 fold), Grp78 (2.0 fold), and calreticulin (3.5 fold) mRNA 60 min post challenge and a significant increase in Grp78 (2.0 fold), calreticulin (2.7 fold) protein levels 2 h postchallenge, relative to fasting levels. The UPR was also activated ex vivo, in human leukocytes cultured in the presence of 15 mmol/l glucose, supporting a specific role for glucose. The oral glucose challenge was associated with a significant increase in the expression of inflammatory cytokines, including interleukin (IL)‐1α/β, IL‐6, and IL‐8, that may result from ER stress. These findings suggest that there is an association between both acute and chronic dysglycemia and ER stress in humans.
Microchip sensors enable rapid, molecular-level profiling of donated lungs for transplant assessment.
BackgroundThe power and simplicity of visual colony counting have made it the mainstay of microbiological analysis for more than 130 years. A disadvantage of the method is the long time required to generate visible colonies from cells in a sample. New rapid testing technologies generally have failed to maintain one or more of the major advantages of culture-based methods.Principal FindingsWe present a new technology and platform that uses digital imaging of cellular autofluorescence to detect and enumerate growing microcolonies many generations before they become visible to the eye. The data presented demonstrate that the method preserves the viability of the microcolonies it detects, thus enabling generation of pure cultures for microbial identification. While visual colony counting detects Escherichia coli colonies containing about 5×106 cells, the new imaging method detects E. coli microcolonies when they contain about 120 cells and microcolonies of the yeast Candida albicans when they contain only about 12 cells. We demonstrate that digital imaging of microcolony autofluorescence detects a broad spectrum of prokaryotic and eukaryotic microbes and present a model for predicting the time to detection for individual strains. Results from the analysis of environmental samples from pharmaceutical manufacturing plants containing a mixture of unidentified microbes demonstrate the method's improved test turnaround times.ConclusionThis work demonstrates a new technology and automated platform that substantially shortens test times while maintaining key advantages of the current methods.
Biomarkers such as proteins and nucleic acids released from human cells, bacteria, and viruses offer a wealth of information pertinent to diagnosis and treatment ranging from cancer to infectious disease. The release of these molecules from within cells is a crucial step in biomarker analysis. Here we show that purely electric-field-driven lysis can be achieved, inline, within a microfluidic channel; that it can produce highly efficient lysis and biomarker release; and, further, that it can do so with minimal degradation of the released biomarkers. Central to this new technology is the use of three-dimensional sharp-tipped electrodes (3DSTEs) in lysis, which we prove using experiment and finite-element modeling produce the electric field concentration necessary for efficient cell wall rupture.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.