Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-07-09
2001-08-21
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S172000, C436S164000, C257S253000, C257S414000, C372S044010, C372S043010, C422S082050, C422S082080, C422S082070, C422S082090, C422S082110, C356S318000
Reexamination Certificate
active
06277651
ABSTRACT:
BACKGROUND OF THE INVENTION
U.S. Pat. No. 5,591,407 describes the use of specific surface sensitive diode laser structures in chemical and biological analysis. In as aspect of that patent the interaction between the laser sensor and the material being sensed is based on the absorption of the material within the effective laser cavity at the surface of the laser. In the present invention, any diode laser may be employed to provide a sensitive chemical or biological detector.
SUMMARY OF THE INVENTION
The finding by LARGENT (1996) Liquid Contact Luminescence, Dissertation, Electrical Engineering Department, University of Florida, Gainesville, Fla. that semiconductor laser wafer material could produce bright optical emission with a liquid contact provides the background to this invention.
The present sensor allows the use of any diode laser structure to be employed to provide a sensitive chemical and biological detector. A diode laser electrochemical sensor is described in U.S. Pat. No. 5,591,407 “Laser Diode Sensor.” The present invention surpasses the capabilities of those sensors by measuring changes in the output power, output spectral characteristics or output signal auto-correlation function to detect the presence of chemical or biological films within the sensitive region.
A diode laser is on top of a substrate. A lasing structure is positioned on top of the substrate. A cap layer is positioned on top of the lasing structure. A segmented electrode is positioned on top of the cap layer. Gaps between the segments of the electrode act as chemical sensors. A sensitive coating is positioned on the cap layer between the segments of the electrode.
In preferred embodiments, passivation at the facets of the diode laser protects the edges from shorting on application of conductive layers. Passivation is accomplished by a layer of electron beam evaporated aluminum oxide. Deposition can be accomplished by laser ablation or magnetron sputtering.
In preferred embodiments, the electrodes and regions between the segmented electrodes are coated by a thin metal island layer. For example, an electron-beam evaporated gold island layer is deposited at thicknesses between 2 nm and 20 nm. Also, the coating containing gold metal islands between the segmented electrodes is modified using cross linked molecules such as (4-succinimidyloxycarbonyl-methyl-a-(2-pyridyldithio)toluene) (SMPT) (spacer arm 2.0 nm) or (m-Maleimidobenzoyl-N-hydrosuccinimide ester) (MBS) (spacer arm 0.99 nm). In this case, the diode laser is first brought into contact with Cleland's reagent (dithiothreitol) (DTT) in excess. Other dithiols include xylyl dithiol, phenyl dithiol, dithiodibenzoic acid, biphenyl dithiol, 1,4-di(4-thiophenylacetylnyl)-2-ethylbenzene (20ADT) and terphenyl dithiol. The laser coated with DTT is then exposed to SMPT or MBS conjugated to a protein of interest. Optimally, the gold coating in the gap region between the contact electrodes should be between 2 nm and 20 nm thick.
In preferred embodiments, a conductive polymer may be deposited in the gap region by electro-deposition or electro polymerization as described U.S. Pat. No. 5,403,451, titled “Method and Apparatus for Pulsed Electrochemical Detection Using Polymer Electroactive Electrodes” to Rivello et al. Poly(pyrrole), poly(thiophene), poly(aniline) or other conducting polymer may be used for this purpose.
A thin film may be used to isolate a sample for testing. In preferred embodiments, the thin film is a capture antibody layer, deposited over a thin oxide layer. The oxide between the contact electrodes is first modified with (3-mercaptopropyl trimethoxysilane) (MPTS). Subsequently, (3-maleimidoprorionic acid) (MPA) followed by 1-ethyl-3-(3-diethylamino propyl)carbodiimide hydrochloride are deposited on the gold layer after the manner suggested by JUNG and WILSON (1996) Polymeric Mercaptosilane-Modified Platinum Electrodes for Elimination of Interference in Glucose Biosensors, Anal. Chem. 68, pp. 591-596. Finally, a 5 microLiter sample of capture antibody solution (1 mg/ml in saline buffer) is brought into contact with the diode laser surface for 24 hours. Similarly, 3-gylcidoxypropyl trimethoxysilane (GPTS) may be deposited directly over the thin oxide layer. The capture antibody may be linked to the oxide layer via N-&ggr;-maleimidobutyryloxy succinimidyl ester (GMBS).
In a preferred embodiment, the thin film in the region between the contact electrodes is deposited by first depositing a thin metal island gold layer (2 nm to 20 nm) and then the laser is allowed to come into contact for 24 hours with a 2% solution of thioctic acid in ethanol. Next, the surface of the diode laser is allowed to dry and then is immersed into a solution of 1% by weight 1-ethyl-3-(3-diethylamino propyl)carbodiimide in acetonitrile for 5 hrs. Finally, a 5 microLiter sample of capture antibody solution (1 mg/ml in saline buffer) is brought into contact with the diode laser surface for 24 hours. The interaction between the laser surface and the capture antibodies in the film is detected as a change in laser output. Changes in output power, output spectral characteristics or output signal auto-correlation function occur where chemical or biological antigens are brought into the vicinity of the surface of the diode laser. The diode laser may also be prepared by deposition of DTT on the laser surface. In a separate reaction, the capture antibody is coupled to SMPT or MBS. Then the DTT-coated laser is coated with antibody-coupled SMPT or MBS. This results in the immobilization of the antibody at the laser surface. Other thiols that may be used to couple the sense molecule to the diode laser surface include xylyl dithiol, phenyl dithiol, dithiodibenzoic acid, biphenyl dithiol, 1,4-di(4-thiophenylacetylnyl)-2-ethylbenzene (20ADT) and terphenyl dithiol. Similarly, the gold coating may be functionalized using isocyanide-based molecules.
In preferred embodiments, the sense film is an oligonucleotide sequence. The sense film is deposited on the diode laser surface using a hybridization solution containing standard saline citrate (SSC), N-lauroylsarcosine, sodium dodecyl sulfate (SDS) and a target deoxyribonucleic acid (DNA) sequence. An oligonucleotide sequence may be deposited at the surface of the diode laser by first coating the diode laser with a thin gold layer. The complimentary oligonucleotide sequence is synthesized with a sulfhydryl group to allow coupling to the laser surface.
A flow cell may be used to bring samples past the diode laser surface where the diode laser is fabricated onto a flow cell. Material flowing in the cell is brought to the surface of the diode laser in the region of the gap. In preferred embodiments, a thin film heater is built into the flow cell to allow denaturing sample DNA and immobilized DNA by heating at 95° C. for 5 minutes or so. The results of hybridization are detected as a change in the output power, output spectral characteristics or output signal autocorrelation function.
Chemical or biological detection is accomplished by monitoring optical output. For this purpose, one or more electrodes may be placed in gap between the segments of the electrode. Two or more additional electrodes may be used to modulate the potential in the gap region.
The modulating electrodes within the gap between the contact electrodes are pulsed in a manner described in JOHNSON and LACOURSE (1990) Liquid Chromatography With Pulsed Electrochemical Detection at Gold and Platinum Electrodes, Anal. Chem., 62 (10), pp. 589a-597a. The potential of one of the two electrodes within the gap region is increased from a starting point of −0.8V to a maximum potential of +0.8V within a short time frame and the output of the diode laser is monitored just after the potential increase. Subsequently, the applied potential of the diode laser gap electrodes from +0.8V to +0.2V is performed, after which time the output signal of the diode laser is monitored.
Where a flow cell is used for sampling, the voltage between gap electrode and contact electrode may
Churchill Russell J.
Coolbaugh Myron T.
Groger Howard P.
Lo K. Peter
Calspan SRL Corporation
Chin Christopher L.
Creighton Wray James
Narasimhan Meera P.
Pham Minh-Quan K.
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