In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.
The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of "electronic taste chips" whereby multiple solution-phase analytes such as acids, bases, metal cations, and biological cofactors were detected and quantitated. The newly fashioned "cardiac chip" exploits a geometry that allows for isolation and entrapment of single polymeric spheres in micromachined pits while providing to each bead the rapid introduction of a series of reagents/washes through microfluidic structures. The combination of these miniaturized components fosters the completion of complex assays with short analysis times using small sample volumes. Optical signals derived from single beads are used to complete immunological tests that yield outstanding assay characteristics. The power and utility of this new methodology is demonstrated here for the simultaneous detection of the cardiac risk factors, C-reactive protein and interleukin-6, in human serum samples. This demonstration represents the first important step toward the development of a useful cardiac chip that targets numerous risk factors concurrently and one that can be customized readily for specific clinical settings.
The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.
The slow development of cost-effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable bio-nano-chip (PBNC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PBNC system employs ≈300 μm-diameter bead sensors composed of agarose “nanonets” that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein-capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead-bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered.
We report on a competitive electrochemical detection system that is free of wash-steps and enables the real-time monitoring of adenosine triphosphate (ATP) in a quantitative manner over a five-log concentration range. The system utilizes a recognition surface based on ATP aptamer (ATPA) capture probes pre-bound to electro-active Flavin adenine dinucleotide (FAD) molecules, and a signaling surface utilizing graphene (Gr) and gold nanoparticle (AuNP) modified carbon paste electrode (Gr-AuNP-CPE) that is optimized to enhance electron transfer kinetics and signal sensitivity. Binding of ATP to ATPA at the recognition surface causes the release of an equivalent concentration of FAD that can be quantitatively monitored in real-time at the signaling surface, thereby enabling a wide linear working range (1.14 ×10−10–3.0×10−5M), a low detection limit (2.01×10−11M using graphene and AuNP modified glassy carbon), and fast target binding kinetics (steady-state signal within 12 minutes at detection limit). Unlike assays based on capture probe-immobilized electrodes, this double-surface competitive assay offers the ability to speed-up target binding kinetics by increasing the capture probe concentration, with no limitations due to intermolecular coulombic interactions and non-specific binding. We utilize the real-time monitoring capability to compute kinetic parameters for target binding and to make quantitative distinctions on degree of base-pair mismatch through monitoring target binding kinetics over a wide concentration range. Based on simplicity of the assay chemistry and the quantitative detection of ATP within fruit and serum media, as demonstrated by comparison of ATP levels against those determined using a standard HPLC-UV absorbance method, we envision a versatile detection platform for applications requiring real-time monitoring over a wide target concentration range.
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