Concentration of analytes
An analyte, concentration technology, applied in the field of detection of analytes, can solve the problems of chemical damage, and replication errors, increasing the cost and complexity of the assay
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Embodiment 1
[0323] In the course of developing embodiments of the technology provided herein, experiments were performed to concentrate small nucleic acids at surfaces for analysis. According to this article and figure 1 The method depicted in a was tested. Briefly, DNA analytes are concentrated in the minority phase of an aqueous two-phase system, from which the analyte is subsequently captured at the surface for analysis (e.g. by single-molecule analysis (e.g. by SiMREPS as described herein)) . In this method, centrifugation is typically performed at approximately 1000 xg for 10-120 minutes in order to provide high capture efficiency of the analyte at the surface of the coverslip. However, experiments performed during the development of the technology presented herein have shown that centrifugation at 1000 xg for approximately 1 minute is sufficient to provide phase separation.
[0324] Furthermore, additional data collected during these experiments indicated that ATPS comprising a m...
Embodiment 2
[0326] During the development of embodiments of the technology provided herein, custom sample wells designed to bring the analyte-rich phase of the ATPS into contact with the capture substrate were produced and tested. In particular, custom-made sample wells were produced that contained a restricted opening at the bottom of the well (e.g. containing approximately 0.01-1 mm 2 area) (see e.g. figure 2 ). A sample well containing a restricted opening provides approximately 0.01-1 mm between the analyte-rich phase of the ATPS and the underlying substrate 2 the contact area. Thus, a sample well containing this restricted opening concentrates the analyte-rich phase in a small area (e.g., approximately 0.01-1 mm 2 ) for efficient surface capture and, in some embodiments, detection of analytes.
Embodiment 3
[0328] During the development of embodiments of the technology provided herein, experiments were conducted to detect analytes with high specificity and single-molecule sensitivity using single-molecule recognition by equilibrium Poisson sampling (SiMREPS) or single-molecule kinetic fingerprinting ( image 3 a to image 3 e).
[0329] The detection method involves heating the sample to denature double-stranded (duplex) DNA and convert it to single-stranded DNA. A brief heat denaturation step is performed in the presence of a high concentration of single-stranded dT10 vector to inhibit re-annealing of complementary analyte strands. Next, single-stranded target DNA is captured by target gene-specific LNA capture probes immobilized on the slide surface. Remaining unbound DNA was washed away prior to analysis using the kinetic fingerprinting method described herein. In these experiments, mutant-specific fluorescent probes were used, optimized for fast association and dissociatio...
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