Simultaneous and rapid colorimetric detection of distinct miRNAs using Split-LAMP.

Introduction: Aberrant microRNA (miRNA) expressions are often discovered in many life threatening diseases such as cancer. In particular, recent studies show combinations of miRNA levels have greater diagnostic accuracy as opposed to single miRNA levels. For point-of-care applications, rapid and sensitive isothermal amplification with loop-mediated isothermal amplification (LAMP) has gained significant interest. Method: We developed a cost-effective point-of-care testing (POCT) device for multiple miRNAs that can integrate miRNA signals into a single output. Results and Discussion: We demonstrate that the loop primers for LAMP can be broken and be used for miRNA detection. This split-LAMP approach provides a logic AND-gate output for two distinct miRNA inputs. We then show that this is potentially useable in point-of-care testing using pH-sensitive dye to give a rapid, colorimetric endpoint readout within 30 min. This novel logic gate approach can potentially be extended to multiple miRNAs such that there can be a powerful diagnostic concept for multiple short RNAs in a point-of-care rapid test.

Floating resistivity detector for microchip electrophoresis.

A newly developed conductivity detector, the floating resistivity detector (FRD), for microchip electrophoresis was introduced in this work. The detector design permits decoupling of the detection circuit from the high separation voltage without compromising separation efficiency. This greatly simplifies the integration of microchip electrophoresis systems. Its method of detection relies on platinum electrodes being dipped in two buffer-filled branched detection probe reservoirs on the microchip device. In this way, analytes passing through the detection window will not pass through and subsequently adsorb onto the electrodes, alleviating problems of electrode fouling due to analyte contamination and surface reactions. A customized microchip design was proposed and optimized stepwise for the new FRD system. Each branched detection probe was determined to be 4.50 mm long with a 0.075 mm detection window gap between them. The distance between the detection window and buffer waste reservoir was determined to be 1.50 mm. The optimized microchip design was subsequently used in the analysis of four groups of analytes - inorganic cations, amino acids, aminoglycosides antibiotics, and biomarkers. Based on the preliminary results obtained, the detection limits were in the range of 0.4-0.7 mg/L for the inorganic cations and 1.5-15 mg/L for the amino compounds.