University of California

Pankaj Singhal

Richard A Mathies

Werner G Kuhr

Alexander N Glazer

Charles A Emrich

Peter Ertl

Jin Xie

Sara Ann Brazill

An electrochemical detector including side channels associated with a separation channel of a sample component separation apparatus is provided. The side channels of the detector, in one configuration, provide a sheath-flow for an analyte exiting the separation channel which directs the analyte to the electrically developed electrochemical detector.

Separation system with a sheath-flow supported electrochemical detector

This invention relates to a microfabricated capillary electrophoresis chip for detecting multiple redox-active labels simultaneously using a matrix coding scheme and to a method of selectively labeling analytes for simultaneous electrochemical detection of multiple label-analyte conjugates after electrophoretic or chromatographic separation.

Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels

A flow-through microchannel (e.g. capillary) biosensor is described for the for the detection of multiple, different analytes (e.g. nucleic acids, proteins, sugars, etc.) targets in a sample by binding them to &ldquo;complementary&rdquo; binding partners (e.g. complementary nucleic acids, ligands, antibodies, etc.). The binding partners are immobilized in different sections of a microchannel (e.g. a fused silica capillary). After fabrication of the biosensor, a sample is flushed through the capillary, and any target analyte(s) contained within the sample are bound to the immobilized binding partner(s) on the microchannel wall forming bound complexes. Finally, the bound complexes are simultaneously denatured along the entire length of the capillary and flushed out past a detector poised downstream, and the analyte concentration is measured (e.g., using sinusoidal voltammetry). Direct electrochemical detection of underivatized DNA is accomplished by oxidizing its sugar backbone and the amine containing nucleobase at the copper electrode. The elution time of the desorbed target DNA(s) is used for the sequence identification of the target. Multiple genetic sequences can be diagnosed by using a single biosensor in this manner. The sensor is highly specific due to hybridization chemistry, and extremely sensitive due to electrochemical detection.

Spatially-encoded analyte detection

Sinusoidal voltammetry was employed to detect both purine and pyrimidine-based nucleic acids. Adenine and cytosine, representing these two classes of nucleic acids, could be detected with nanomolar detection limits at a copper electrode under these conditions, where the sensitivity for adenine was much higher than that for cytosine. Detection limits for purine-containing nucleotides (e.g., adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP)) were on the order of 70-200 nM using this method. These detection limits are achieved for native nucleotides and are over two orders of magnitude lower than those found with UV absorbance detection. Pyrimidine-based nucleotides could also be detected with high sensitivity due to the presence of a sugar backbone which is electroactive at the copper surface. This detector is not fouled by the nucleotides and can be used for the sensitive detection of analyses eluting continuously from a chromatography column or a electrophoresis capillary. Entire nucleic acid molecules can be readily detected. For example, both single stranded and double stranded DNA is detected with a detection limit in the picomolar concentration range (i.e., 10.sup.-12 moles/L). In the present invention, the signal from a double stranded DNA is roughly twice that arising from a single stranded DNA strand of the same length.

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Pasadena, CA, United States of America

Pankaj Singhal