Advances in materials chemistry offer a range of nanostructured shapes and textures for building new biosensors. Previous reports have implied that controlling the properties of sensor substrates can improve detection sensitivities, but the evidence remains indirect. Here we show that by nanostructuring the sensing electrodes, it is possible to create nucleic acid sensors that have a broad range of sensitivities and that are capable of rapid analysis. Only highly branched electrodes with fine structuring attained attomolar sensitivity. Nucleic acid probes immobilized on finely nanostructured electrodes appear more accessible and therefore complex more rapidly with target molecules in solution. By forming arrays of microelectrodes with different degrees of nanostructuring, we expanded the dynamic range of a sensor system from two to six orders of magnitude. The demonstration of an intimate link between nanoscale sensor structure and biodetection sensitivity will aid the development of high performance diagnostic tools for biology and medicine.
The use of single wall carbon nanotubes (SWCNTs) in current and future applications depends on the ability to process SWCNTs in a solvent to yield high-quality dispersions characterized by individual SWCNTs and possessing a minimum of SWCNT bundles. Many approaches for the dispersion of SWCNTs have been reported. However, there is no general assessment which compares the relative quality and dispersion efficiency of the respective methods. Herein we report a quantitative comparison of the relative ability of "wrapping polymers" including oligonucleotides, peptides, lignin, chitosan, and cellulose and surfactants such as cholates, ionic liquids, and organosulfates to disperse SWCNTs in water. Optical absorption and fluorescence spectroscopy provide quantitative characterization (amount of SWCNTs that can be suspended by a given surfactant and its ability to debundle SWCNTs) of these suspensions. Sodium deoxy cholate (SDOCO), oligonucleotides (GT)(15), (GT)(10), (AC)(15), (AC)(10), C(10-30), and carboxymethylcellulose (CBMC-250K) exhibited the highest quality suspensions of the various systems studied in this work. The information presented here provides a good framework for further study of SWCNT purification and applications.
Detection of biomolecules at low abundances is crucial to the rapid diagnosis of disease. Impressive sensitivities, typically measured with small model analytes, have been obtained with a variety of nano- and microscale sensors. A remaining challenge, however, is the rapid detection of large native biomolecules in real biological samples. Here we develop and investigate a sensor system that directly addresses the source of this challenge: the slow diffusion of large biomolecules traveling through solution to fixed sensors, and inefficient complexation of target molecules with immobilized probes. We engineer arrayed sensors on two distinct length scales: a ∼100 μm length scale commensurable with the distance bacterial mRNA can travel in the 30 min sample-to-answer duration urgently required in point-of-need diagnostic applications; and the nanometer length scale we prove necessary for efficient target capture. We challenge the specificity of our hierarchical nanotextured microsensors using crude bacterial lysates and document the first electronic chip to sense trace levels of bacteria in under 30 min.
The analysis of panels of nucleic acid biomarkers offers valuable diagnostic and prognostic information for cancer management. A cost-effective, highly sensitive electronic chip would offer an ideal platform for clinical biomarker readout and would have maximal utility if it was (i) multiplexed, enabling on-chip assays of multiple biomarkers, and (ii) able to perform direct (PCR-free) readout of disease-related genes. Here we report a chip onto which we integrate novel nanostructured microelectrodes and with which we directly detect cancer biomarkers in heterogeneous biological samples-both cell extracts and tumor tissues. Coarse photolithographic microfabrication defines a multiplexed sensing array; bottom-up fabrication of nanostructured microelectrodes then provides sensing elements. We analyzed a panel of mRNA samples for prostate cancer related gene fusions using the chip. We accurately identified gene fusions that correlate with aggressive prostate cancer and distinguished these from fusions associated with slower-progressing forms of the disease. The multiplexed nanostructured microelectrode integrated circuit reported herein provides direct, amplification-free, sample-to-answer in under 1 h using the 10 ng of mRNA readily available in biopsy samples.
We report an electrochemical nucleic acids sensing system that exhibits high sensitivity and specificity when challenged with heterogeneous samples of RNA. The platform directly detects specific RNA sequences in cellular and clinical samples without any sample labeling or PCR amplification. The sensor features an electrode platform consisting of three-dimensional gold nanowires, and DNA or RNA hybridization is detected using an electrocatalytic reporter system. In this study, probes made of peptide nucleic acid (PNA) are used to detect a newly identified cancer biomarkera gene fusion recently associated with prostate cancer. The system is able to detect the fusion sequence with 100 fM sensitivity, and retains high sensitivity even in the presence of a large excess of non-complementary sequences. Moreover, the sensor is able to detect the fusion sequence in as little as 10 ng of mRNA isolated from cell lines or 100 ng total RNA from patient tissue samples. The PNA-nanowire nucleic acids sensor described is one of the first electrochemical sensors to directly detect specific mRNAs in unamplified patient samples.
A chip‐based platform is reported that is able to detect as few as 10 cancer cells. By developing sub‐milliscale sensors that are able to capture slow moving biological targets with high efficiency (see picture; scale bar 50 μm), cancer‐specific sequences were detected in crude lysates of leukemia cells. This achievement relied on the development of a new type of molecular probe that improves the solubility and performance of neutral nucleic acids.
An important goal for improved diagnosis and management of infectious disease is the development of rapid and accurate technologies for the decentralized detection of bacterial pathogens. Most current clinical methods that identify bacterial strains require time-consuming culture of the sample or procedures involving the polymerase chain reaction. Neither of these approaches has enabled testing at the point-of-need because of the requirement for skilled technicians and laboratory facilities. Here, we demonstrate the performance of an effective, integrated platform for the rapid detection of bacteria that combines a universal bacterial lysis approach and a sensitive nanostructured electrochemical biosensor. The lysis is rapid, is effective at releasing intercellular RNA from bacterial samples, and can be performed in a simple, cost-effective device integrated with an analysis chip. The platform was directly challenged with these unpurified lysates in buffer and urine. We successfully detected the presence of bacteria with high sensitivity and specificity and achieved a sample-to-answer turnaround time of 30 min. We have met the clinically relevant detection limit of 1 cfu/μL, indicating that uncultured samples can be analyzed. This advance will greatly reduce time to successful detection from days to minutes.
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