Automated computer controlled reporter device for conducting imunnoassay and molecular biology procedures

• US 6,562,209 B1
• Filed: 04/19/2001
• Issued: 05/13/2003
• Est. Priority Date: 04/19/2001
• Status: Expired due to Term

• Current Assignee: Northrop Grumman Systems Corporation (Northrop Grumman Corporation)
• Original Assignee: Northrop Grumman Corporation
• Inventors: Zsolnay, Denes L., McHugh, Richard K., MacPhee, Robert D. Jr., Sullivan, Brian M.
• Primary Examiner(s): Warden, Sr., Robert J.
• Assistant Examiner(s): Olsen, Kaj K.
• Application Number:
  US09/837,946
• Time in Patent Office: 754 Days
• Field of Search: 204/403.01, 204/403.14, 435/817, 435/286.1, 435/287.1
• US Class Current: 204/403.01
• CPC Class Codes: G01N 2035/1025 Fluid level sensing
• CPC Class Codes: G01N 27/48 using polarography, i.e. measuring changes in current under a slowly-varying voltage
• CPC Class Codes: G01N 35/00594 Quality control, including calibration or testing of components of the analyser
• CPC Class Codes: G01N 35/00693 Calibration
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: G01N 35/08 using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

An electronic controller, such as a programmed microcontroller (33), controls a series of pumps (P1-P6) to automatically sequence the pumping of the individual fluids required by the ELISA procedure into a cell (or cells) necessary to produce a reporter, control the positioning of the carrier of the reporter (32, 35) adjacent the reporter sensor (28), analyze the data obtained from the reporter sensor and display (36) the concentration of the bioagent determined from the analysis of the foregoing data. The foregoing elements automate the ELISA procedure. Once started, the apparatus, governed by the program, conducts the test automatically without the necessity for human intervention. In accordance with a specific aspect, the foregoing components may be housed in a single package for easy portability; may be battery powered or line powered.

Claims

1. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising;

a plurality of vessels, said plurality of vessels including;

a first vessel containing 1°

antibody coated magnetic beads in liquid;

a second vessel containing a wash solution;

a third vessel containing a 2°

antibody linked enzyme;

a fourth vessel containing a substrate reporter; and

sample holding means containing a sample solution containing said bioagent, protein or nucleic acid;

an examination cell;

an electronic controller, said electronic controller including a program, a start switch and a display;

a sensor for electrically detecting the level of reporter present in said examination cell and supplying said detected level of reporter present to said electronic controller;

said electronic controller for motivating passage of the respective contents of each of said vessels into said examination cell when required by said program and for motivating removal of the contents of said examination cell in whole or in part when required by said program;

said program configured for performing an ELISA technique, wherein motivation of the contents of each of said vessels is motivated in a sequence;

said program including means for motivating the contents of said fourth vessel into said examination cell and initiating collection of the detected level of reporter present at said sensor at each of a plurality of time intervals following motivation of the contents of said fourth vessel into said examination cell;

whereby insertion of the contents of said fourth vessel into said examination cell when said respective bioagent, protein or nucleic acid is present in said examination cell produces an electrochemical redox recycling reaction inside said examination cell to produce levels of reporter that increases with time;

said program further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent, protein or nucleic acid present in said sample when said respective bioagent, protein or nucleic acid is present in said sample and displaying said concentration on said display.

2. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid comprising as defined in claim 1, wherein said electronic controller includes a look-up table, said look-up table containing a plurality of numbers defining slopes of lines representing detected levels of reporter versus time and a plurality of bioagent concentrations with each of said plurality of bioagent concentrations being associated with a respective one of said plurality of numbers, wherein for each slope represented in said look-up table, a concentration of said respective bioagent, protein or nucleic acid may be determined;

wherein said analysis program further comprises;

a regression analysis program for performing a least-square linear regression analysis on said detected level of reporter taken at each of said plurality of time intervals to determine a number, said number defining a slope of a line representing detected levels of reporter versus time;

a look-up program for looking up said number determined by said regression analysis program in said look-up table and locating the corresponding concentration of said bioagent represented thereby.

3. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 1, further comprising:

a plurality of electric pumps, each pump being associated with a respective one of said first through fourth vessels and said sample holding means for conveying contents from the respective vessels into said examination cell when said pump is energized;

said electronic controller being coupled to said plurality of pumps for controlling the energization of said pumps in accordance with said controller program.

4. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 1, further comprising:

an aspirating pump, said aspirating pump for pumping contents from said examination cell when energized by said electronic controller.

5. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising:

a plurality of vessels, said plurality of vessels including;

a first vessel containing 1°

antibody coated magnetic beads in liquid;

a second vessel containing a wash solution;

a third vessel containing a 2°

antibody linked enzyme;

a fourth vessel containing a substrate reporter; and

sample holding means containing a sample solution containing said bioagent, protein or nucleic acid;

an examination cell;

an electronic controller, said electronic controller including a program, a start switch and a display;

a sensor for electrically detecting the level of reporter present in said examination call and supplying said detected level of reporter present to said electronic controller;

said electronic controller for motivating passage of the respective contents of each of said vessels into said examination cell when required by said program and for motivating removal of the contents of said examination cell in whole or in part when required by said program;

said program configured for performing an ELISA technique, wherein motivation of the contents of each of said vessels is motivated in a sequence to pass the contents of the respective vessels into said examination cell;

said program including means for motivating the contents of said fourth vessel into said examination cell and initiating collection of the detected level of reporter present at said sensor at each of a plurality of time intervals following motivation of the contents of said fourth vessel into said examination cell;

whereby insertion of the contents of said fourth vessel into said examination cell when said respective bioagent, protein or nucleic acid is present in said examination cell produces an electrochemical redox recycling reaction inside said examination cell to produce levels of reporter that increases with time;

said program further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent, protein or nucleic acid present in said sample when said respective bioagent, protein or nucleic acid is present in said sample and displaying said concentration on said display; and

further comprising;

a magnet controlled by said electronic controller for producing a magnetic field inside said examination cell when required by said program; and

means for washing the fluid in said examination cell;

said means for washing the fluid including;

first means for motivating said magnet to produce a magnetic field in said examination cell responsive to a command from said electronic controller, wherein magnetic beads in said examination cell are drawn toward said magnet leaving a portion of said examination cell vacant of magnetic beads;

second means for aspirating fluid from said bead vacated region of said examination cell when said magnetic field is present responsive to a command from said electronic controller, and

third means for motivating wash fluid from said second vessel into said examination cell responsive to a command by said electronic controller following aspiration of fluid by said second means.

6. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising:

a plurality of vessels, said plurality of vessels including;

a first vessel containing 1°

antibody coated magnetic beads in liquid;

a second vessel containing a wash solution;

a third vessel containing a 2°

antibody linked enzyme;

a fourth vessel containing a substrate reporter; and

sample holding means containing a sample solution containing said bioagent, protein or nucleic acid;

an examination cell;

an electronic controller, said electronic controller including a program, a start switch and a display;

a sensor for electrically detecting the level of reporter present in said examination cell and supplying said detected level of reporter present to said electronic controller;

said electronic controller for motivating passage of the respective contents of each of said vessels into said examination cell when required by said program and for motivating removal of the contents of said examination cell in whole or in part when required by said program;

said electronic controller including a look-up table, said look-up table containing a plurality of numbers defining slopes of lines representing detected levels of reporter versus time and a plurality of bioagent concentrations with each of said plurality of bioagent concentrations being associated with a respective one of said plurality of numbers, wherein for each slope represented in said look-up table, a concentration of said respective bioagent, protein or nucleic acid may be determined;

said program configured for performing an ELISA technique, wherein motivation of the contents of each of said vessels is motivated in a sequence to pass the contents of the respective vessels into said examination cell;

said program including means for motivating the contents of said fourth vessel into said examination cell and initiating collection of the detected level of reporter present at said sensor at each of a plurality of time intervals following motivation of the contents of said fourth vessel into said examination cell;

whereby insertion of the contents of said fourth vessel into said examination cell when said respective bioagent, protein or nucleic acid is present in said examination cell produces an electrochemical redox recycling reaction inside said examination cell to produce levels of reporter that increases with time;

said program further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent, protein or nucleic acid present in said sample when said respective bioagent, protein or nucleic acid is present in said sample and displaying said concentration on said display.

said analysis program further including;

a regression analysis program for performing a least-square linear regression analysis on said detected level of reporter taken at each of said plurality of time intervals to determine a number, said number defining a slope of a line representing detected levels of reporter versus time; and

a look-up program for looking up said number determined by said regression analysis program in said look-up table and locating the corresponding concentration of said bioagent represented thereby; and

further comprising;

a magnet controlled by said electronic controller for producing a magnetic field inside said examination cell when required by said program; and

means for washing the fluid in said examination cell;

said means for washing including;

first means for motivating said magnet to produce a magnetic field in said examination cell responsive to a command from said electronic controller, wherein magnetic beads in said examination cell are drawn toward said magnet leaving a portion of said examination cell vacant of magnetic beads;

second means for aspirating fluid from said bead vacated region of said examination cell when said magnetic field is present responsive to a command from said electronic controller, and

third means for motivating wash fluid from said second vessel into said examination cell responsive to a command by said electronic controller following aspiration of fluid by said second means.

7. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 6, wherein said means for motivating the contents of said fourth vessel into said examination cell and initiating assembly of the detected level of reporter present at each of a plurality of time intervals from said sensor further includes means for motivating said magnet to produce a magnetic field in said examination cell that extends through said sensor, wherein magnetic beads in said examination tube are drawn to said sensor.

8. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid comprising as defined in claim 7, wherein said sensor produces an electrical current that is proportional to the quantity of reporter present in said examination cell, whereby the electrical current level increases as the amount of reporter developed with time in said examination cell increases.

9. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 8, wherein said sensor comprises a pair of spaced comb-like shaped electrodes interdigitally arranged, said spaced electrodes being located inside said examination cell to provide a current carrying path in a gap between said electrodes through at least a portion of the contents of said examination cell, and a pair of electrical conductors for respectively connecting each of said spaced electrodes to a source of potential external to said examination cell.

10. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 9, further comprising:

a plurality of electric pumps, each pump being associated with a respective one of said first through fourth vessels and said sample holding means for conveying contents from the respective vessel into said examination cell when said pump is energized;

said electronic controller being coupled to said plurality of pumps for controlling the energization of said pumps in accordance with said controller program.

11. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising:

a plurality of vessels, said plurality of vessels including;

a first vessel for holding 1°

antibody coated magnetic beads in liquid;

a second vessel for holding a wash solution;

a third vessel for holding a 2°

antibody linked enzyme;

a fourth vessel for holding a substrate reporter; and

sample holding means for holding a sample solution containing said bioagent, protein or nucleic acid;

an examination cell;

a plurality of electric pumps, each pump being associated with a respective one of said first through fourth vessels and said sample holding means for conveying contents from the respective vessel into said examination cell when said pump is energized;

an electronic controller, said electronic controller including a program, a start switch and a display;

said electronic controller being coupled to said plurality of pumps for controlling the energization of said pumps in accordance with said controller program;

said electronic controller further including a look-up table, said look-up table containing a plurality of numbers defining slopes and a plurality of bioagent concentrations with each of said plurality of bioagent concentrations being associated with a respective one of said plurality of numbers, wherein for each slope represented in said look-up table, a concentration of said bioagent may be determined;

an aspirating pump, said aspirating pump for pumping contents from said examination cell when energized by said electronic controller;

a magnet controlled by said electronic controller for producing a magnetic field inside said examination cell when required by said program;

a sensor for electrically detecting the level of reporter present in said examination call and supplying said detected level of reporter present to said electronic controller, said sensor for producing an electrical current that is proportional to the quantity of reporter present in said examination cell;

said sensor further comprising a pair of spaced comb-like shaped electrodes interdigitally arranged, said spaced electrodes being located inside said examination cell to provide a current carrying path in a gap between said electrodes through at least a portion of the contents of said examination cell, and a pair of electrical conductors for respectively connecting each of said spaced electrodes to a source of potential external to said examination cell

said electronic controller for motivating passage of the respective contents of each of said vessels into said examination cell when required by said program and for motivating removal of the contents of said examination cell in whole and/or in part when required by said program;

said program further configured for performing an ELISA technique, wherein motivation of the contents of each of said vessels is performed in a sequence to pass contents of the respective vessel into said examination cell;

means for washing the fluid in said examination cell, said means for washing further comprising;

first means for motivating said magnet to produce a magnetic field in said examination cell responsive to a command from said electronic controller, wherein magnetic beads in said examination cell are drawn toward said magnet leaving a portion of said examination cell vacant of magnetic beads;

second means for aspirating fluid from said bead vacated region of said examination cell when said magnetic field is present responsive to a command from said electronic controller, and

third means for motivating wash fluid from said second vessel into said examination cell responsive to a command by said electronic controller following aspiration of fluid by said second means;

said program including means for motivating the contents of said fourth vessel into said examination cell, motivating said magnet to produce a magnetic field in said examination cell that extends through said sensor to draw magnetic beads in said examination tube to said sensor, and for obtaining from said sensor the detected level of reporter present at each of a plurality of time intervals at said sensor;

whereby insertion of the contents of said fourth vessel into said examination cell when said bioagent is present in said examination cell produces an electrochemical reaction inside said examination cell to produce levels of reporter that increases with time;

said program further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent present in said sample when said bioagent is present in said sample and displaying said concentration on said display;

said analysis program including;

a regression analysis program for performing a least-square linear regression analysis on said detected level of reporter taken at each of said plurality of time intervals to determine a number, said number defining a slope of a line representing detected levels of reporter versus time;

a look-up program for looking up said number determined by said regression analysis program in said look-up table and locating the corresponding concentration of said bioagent represented thereby.

12. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising:

an examination cell;

an electronic controller, said electronic controller including a programmed microprocessor, a start switch and a display;

a sensor for electrically detecting the level of reporter present in said examination cell and supplying said detected level of reporter present to said electronic controller;

control means, controlled by said electronic controller, for automatically producing a primary/analyte/secondary antibody with enzyme linking complex (1°

Ab/analyte/2°

Ab-enz complex) attached to magnetic beads and depositing said complex in said examination cell and for subsequently depositing a substrate in said examination cell;

said electronic controller for obtaining from said sensor the detected level of reporter present at said sensor at each of a plurality of time intervals;

whereby insertion of said substrate into said examination cell when said bioagent, protein or nucleic acid is present in said examination cell produces an electrochemical redox recycling reaction inside said examination cell to produce levels of reporter that increases in level with the passage of time;

said electronic controller further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent present in said sample when said bioagent, protein or nucleic a acid is present in said sample and displaying said concentration on said display.

13. The apparatus for conducting an electrochemical ELISA for a bioagent, protein or nucleic acid as defined in claim 12, wherein said substrate comprises:

p-amino phenyl galactopyranoside (PAP-GP).

14. The apparatus for conducting an electrochemical ELISA for a bioagent, protein and/or nucleic acid comprising as defined in claim 12, wherein said electronic controller includes a look-up table, said look-up table containing a plurality of numbers defining slopes of lines that represent detected levels of reporter versus time and a plurality of respective bioagent, protein or nucleic acid concentrations with each of said plurality of bioagent, protein or nucleic acid concentrations being associated with a respective one of said plurality of numbers, wherein for each slope of a line represented in said took-up table, a concentration of said bioagent, protein or nucleic acid may be determined;

and

wherein said analysis program further comprises;

a regression analysis program for performing a least-square linear regression analysis on said detected level of reporter taken at each of said plurality of time intervals to determine a number, said number defining a slope of a line representing detected levels of reporter versus time;

a look-up program for looking up said number determined by said regression analysis program in said look-up table and locating the corresponding concentration of said bioagent represented thereby.

15. Apparatus for conducting an electrochemical ELISA for a bioagent, protein and/or nucleic acid as defined in claim 12, wherein said apparatus includes:

sample holding means for holding a sample in a solution, said sample for examination being a bioagent, protein or nucleic acid;

a second vessel for holding 1°

antibody coated magnetic beads in liquid;

a third vessel for holding a 2°

antibody-enzyme;

a fourth vessel for holding a wash solution;

a plurality of pumps, each of said plurality of pumps being associated with a respective one of said sample holding means and said second through fourth vessels;

a reaction cell;

a plurality of fluid conduits, each of said plurality of fluid conduits being associated with a respective one of said plurality of pumps for passing fluid pumped by an associated pump from the respective sample holding means and second through fourth vessels into said reaction cell;

a fifth vessel for holding a p-aminophenyl-glalactopyranoside (PAP-GP) in a liquid solution;

an electric two way valve, said valve having a normally closed inlet, a normally open inlet and an outlet;

an aspirating conduit coupled between said reaction cell and said normally closed inlet of said two-way valve;

a drain conduit coupled between said fifth vessel and said normally open inlet of said two-way valve;

a transfer conduit coupled between said outlet of said two-way valve and said examination cell;

a second drain conduit coupled to said examination cell;

an aspirating electric pump coupled to said second drain conduit, whereby operation of said pump produces an aspirating force in said examination cell, wherein operation of said pump when said two way valve is not energized draws fluid from said reaction cell into said examination cell and wherein operation of said pump when said two way valve is energized draws said PAP-GP from said fifth vessel into said examination cell.

16. Apparatus for conducting an electrochemical ELISA for a bioagent, protein and/or nucleic acid as defined in claim 12 wherein said apparatus includes:

sample holding means for holding a sample in a solution, said sample for examination as a bioagent;

a second vessel for holding 1°

antibody coated magnetic beads in liquid;

a third vessel for holding a 2°

antibody-enzyme;

a fourth vessel for holding a wash solution;

a fifth vessel for holding a p-aminophenyl-glalactopyranoside (PAP-GP) in a liquid solution;

a plurality of pumps, each of said plurality of pumps being associated with a respective one of said second through fifth vessels and said sample holding means;

a plurality of fluid conduits, each of said plurality of fluid conduits being associated with a respective one of said plurality of pumps for passing fluid pumped by an associated pump from the respective second through fifth vessels and said sample holding means into said examination cell when required by said electronic controller.

17. Apparatus for conducting an electrochemical enzyme linked immunosorbent assay (ELISA) for a bioagent, protein or nucleic acid comprising:

an examination cell;

an electronic controller, said electronic controller including a programmed microprocessor, a start switch and a display;

a sensor for electrically detecting the level of reporter present in said examination cell and supplying said detected level of reporter present to said electronic controller;

control means, controlled by said electronic controller, for automatically producing a primary/analyte/secondary antibody with enzyme linking complex (1°

Ab/analyte/2°

Ab-enz complex) attached to magnetic beads and depositing said complex in said examination cell and for subsequently depositing a substrate in said examination cell;

said electronic controller for obtaining from said sensor the detected level of reporter present at said sensor at each of a plurality of time intervals;

whereby insertion of said substrate into said examination cell when said bioagent, protein or nucleic is present in said examination cell produces an electrochemical redox recycling reaction inside said examination cell to produce levels of reporter that increases in level with the passage of time;

said electronic controller further including an analysis program for analyzing the detected level of reporter at each of said plurality of time intervals and determining the concentration of bioagent present in said sample when said bioagent, protein or nucleic acid is present in said sample and displaying said concentration on said display; and

further comprising;

a magnet controlled by said control means for producing a magnetic field inside said examination cell when required by said electronic controller; and

wherein said control means includes washing means for washing the fluid in said examination cell;

said washing means including;

first means for motivating said magnet to produce a magnetic field in said examination cell responsive to a command from said electronic controller, wherein magnetic beads in said examination cell are drawn toward said magnet leaving a portion of said examination cell vacant of magnetic beads;

second means for aspirating fluid from said magnetic bead vacated portion of said examination cell when said magnetic field is present responsive to a command from said electronic controller, and

third means for motivating wash fluid from said second vessel into said examination cell responsive to a command by said electronic controller following aspiration of fluid by said second means.