CN102520161A - Biosensor chip, kit and detection method for SPR (Surface Plasmon Resonance) dual-channel method to detect specific prostate antigen - Google Patents

Biosensor chip, kit and detection method for SPR (Surface Plasmon Resonance) dual-channel method to detect specific prostate antigen Download PDF

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CN102520161A
CN102520161A CN2012100019653A CN201210001965A CN102520161A CN 102520161 A CN102520161 A CN 102520161A CN 2012100019653 A CN2012100019653 A CN 2012100019653A CN 201210001965 A CN201210001965 A CN 201210001965A CN 102520161 A CN102520161 A CN 102520161A
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psa
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antigen
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向娟
彭贞
陈胜华
陈晓青
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Central South University
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Abstract

The invention discloses a biosensor chip, a kit and a detection method for an SPR (Surface Plasmon Resonance) dual-channel method to detect a specific prostate antigen. The method and the biosensor chip disclosed by the invention can be applied to synchronous quantitative detection of f-PSA and t-PSA in a sample with a detection limit reaching 50pg/ml, and can be used for effectively increasing the accuracy of a f/t value so as to serve as a reference for distinguishing the prostate cancer and the benign prostatic hyperplasia. The chip, the kit and the detection method, disclosed by the invention, have the advantages of high selectivity, low detection limit, good repeatability, wide application prospect and the like.

Description

A kind of bio-sensing chip, the kit that SPR bilateral Dow process detects PSA, detection method of being used for
Technical field
The invention belongs to prostate cancer detectable field.Concrete, relate to a kind of with surface plasma resonance technology (Surface Plasmon Resonance, the SPR) method of detection PSA, and the SPR bio-sensing chip that adopts this method, kit,
Background technology
It is one of major technique means of cancer screening, diagnosis and prognosis that tumor markers detects.PSA is a kind of protease that is mainly produced by prostate epithelial cell, is to have high tissue and organ specificity, the tumor markers of high susceptibility.Under normal physiological conditions, PSA is secreted in the seminal fluid through conduit, its concentration ratio in seminal fluid in serum concentration exceed 1,000,000 times.Between prostatic acinus and catheter lumen and the blood circulation system; Exist and organize barrier; When suffering from prostate cancer, this one deck natural cover can cause destruction because of the growth of the madness of tumour cell, and PSA will penetrate into the rising significantly that causes blood-serum P SA concentration in the blood in a large number.In view of PSA has strict histoorgan and cell-specific, at present clinical mainly to measure the main means of PSA as screening prostate cancer and hyperplasia of prostate.
But the tumour-specific of PSA is relatively poor.With age, extraneous factor etc. all might cause PSA concentration to raise; Particularly benign prostatic hyperplasis and prostatitis all can cause the rising of t-PSA (being total PSA) concentration; When t-PSA concentration is in 4-10ng/ml; Prostate cancer and benign prostatic hyperplasis all have the higher incidence of disease, claim that traditionally this zone is the diagnosis gray area.In order to improve the specificity of gray area, reduce the misdiagnosis rate of prostate cancer, people have proposed several different methods it have been detected, comprising PSA density, PSA gather way, references such as prostate transitional zone density, age or ethnic specific factors.Discovered in recent years detects f-PSA (being free state PSA), and relatively the ratio (f/t) of f-PSA/t-PSA can effectively be distinguished hyperplasia of prostate and prostate cancer, particularly in the diagnosis grey area, can improve the diagnosis authenticity effectively, reduces misdiagnosis rate.
At present; The large-scale instrument that the method for PSA is based on large-scale testing agency in traditional detection serum detects; Though these methods can detect super low concentration, shortcomings such as detection method is loaded down with trivial details, expensive, used detectable is many, sense cycle is long are arranged.Given this, exploitation a kind of can be fast, accurately, the unusual necessity of the low detection means of expense.Along with the continuous development of immunosensor, use the core component of the material of nano particle and nanostructured as immunosensor, make up the PSA immunosensor, thereby the clinical fast detecting that has realized PSA becomes possibility.Till now; The detection method of having reported mainly comprises ELISA (ELISA), the nano particle method of immunochromatographic method, time resolution immunofluorescence technique, chemiluminescence immunoassay method, SPR method, fluorescence immunoassay method, SERS method, electrochemical method, biological coding, QCM method, microcantilever method etc.In said method, the immunochromatographic method expense is the cheapest, also very convenient, but its topmost shortcoming is that sensitivity is low, can only realize sxemiquantitative.Radio immunoassay has higher sensitivity, accuracy and degree of accuracy, but reagent exists radioactive contamination and label half life period weak point.And the chemiluminescence immunoassay method all needs import at present instrument of China and reagent, and it is higher to detect cost.
The SPR technology is a kind of optical detective technology based on the chip surface variations in refractive index, have fast, sensitive, exempt from advantage such as mark, but real-time online ground follow the tracks of solid-liquid interface and react, having broad application prospects aspect the biomolecule detection such as albumen and nucleic acid.But traditional SPR detection method is difficult to detect the biomolecule of low-molecular-weight, super low concentration.The concentration of f-PSA in human serum is very low, directly utilizes traditional SPR technology to be difficult to detect.Density is big, specific inductive capacity big, excellent biological compatibility and nm of gold has, and can strengthen spr signal greatly.Given this, we combine both advantages, design the SPR technology for detection super low concentration f-PSA that amplifies based on nm of gold, in conjunction with SPR binary channels detection technique, obtain f/t synchronously.
Summary of the invention
One of the object of the invention is: a kind of SPR biochip and kit that PSA detects that be used for is provided; Two of the object of the invention is: the SPR binary channels detection method that a kind of PSA is provided.Through the present invention, can the reduced sample treatment step, utilize high flux, high sensitive, the high special characteristic of biochip technology, overcome the deficiency of existing detection method.
A kind of bio-sensing chip that is used for SPR bilateral Dow process detection PSA; Matrix is glass sheet; Glass sheet surface is coated with golden film; There is the mercaptan carboxylic acid's class unimolecular layer that forms through chemical modification on gold film surface, and the zone corresponding to two passages in the SPR binary channels on the unimolecular layer is fixed with f-PSA antibody and t-PSA antibody respectively, and seals remaining monomolecular avtive spot with aminoacetic acid.
Described golden film thickness is 40-60nm.
Described mercaptan carboxylic acid's class is that methyl unit's number is the HS (CH of 8-16 2) nCOOH.
Go back spraying plating between glass sheet and the golden film the thick chromium of one deck 2nm is arranged.
A kind of kit that is used for SPR bilateral Dow process detection PSA comprises described bio-sensing chip, t-PSA antigen and Au mark f-PSA antigen.
Described Au mark f-PSA antigen is to mix with excessive f-PSA antigenic solution, refrigerated centrifuge, obtain after removing supernatant liquor through nm of gold; Wherein said Au diameter of nano particles is 15-40nm.
The method that a kind of PSA detects may further comprise the steps:
(1) described bio-sensing chip is placed in the SPR binary channels, corresponding two sense channels of band antibody regions difference on the chip mark among the normal concentration f-PSA antigen flow injection process SPR Au to f-PSA detection of antibodies passage should be arranged; Normal concentration t-PSA antigen flow injection through among the SPR to t-PSA detection of antibodies passage should be arranged, and record spr signal production standard curve;
(2) with two sense channels of pretreated testing sample flow injection process SPR, and the record spr signal changes;
(3) continue to mark f-PSA antigen, and the record spr signal changes to the passage that f-PSA antibody should be arranged is injected Au;
(4) according to standard working curve, confirm that the spr signal of above-mentioned different passages changes corresponding respectively f-PSA and t-PSA concentration, obtain the f/t value;
Described Au mark f-PSA antigen is to mix with excessive f-PSA antigenic solution, refrigerated centrifuge, obtain after removing supernatant liquor through nm of gold; Wherein said Au diameter of nano particles is 15-40nm.
Each normal concentration antigen, testing sample all is 5-50 μ l/min with the flowing velocity of gold mark antigen.
Prepare SPR bio-sensing chip of the present invention, referring to Fig. 1, method may further comprise the steps:
(1) glass sheet after will handling totally is put into vacuum magnetic control and spatters the thick chromium of spraying plating one deck 2nm on the sputtered films of bismuth machine, on the chromium film, continues the golden film of spraying plating one deck 40-60nm subsequently.
(2) use the HS (CH of methyl unit's number as 8-16 2) nThe alcoholic solution of COOH soaks golden film, forms mercaptan carboxylic acid's class unimolecular layer.
(3) with the mixed liquor activation mercaptan carboxylic acid class of 1-(3-dimethylamine propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-carboxyl thiosuccimide (NHS) terminal-COOH;
(4) injection Anti-PSA is connected on the golden film it; Zone corresponding to two passages in the SPR binary channels is fixed with f-PSA antibody and t-PSA antibody respectively;
(5) with ethylaminoethanol confining surface residual activity site, promptly make said SPR biochip.
The employing binary channels detects, and the instrument principle of work promptly adopts the SPR response of two autonomous channels of method synchronous detection of two light sources, dual-detector referring to Fig. 3.
The advantage and the effect of present technique invention are following:
(1) the present invention adopts Au mark PSA antigen, has improved the sensitivity that detects, and has reduced the least concentration that detects;
(2) this SPR binary channels detection method has reduced the processing requirements of sample, has simplified operation steps, has shortened detection time;
(3) the present invention does not need expensive detecting instrument, has reduced the use cost and the experiment condition of chip.
In a word, this technology succeeds in developing the high flux that will really realize chip technology, special, sensitive, characteristics fast.This technology is that the diagnosis of prostate cancer, the tracking and the prognosis of therapeutic process provide technical support and reference, for the prevention and the treatment of prostate cancer provides important directive function.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is further specified, and can not limit the present invention.
Fig. 1. the SPR bio-sensing chip synoptic diagram that designs for the present invention;
Fig. 2. for PSA SPR binary channels of the present invention detects schematic diagram;
Fig. 3. be binary channels SPR detecting instrument fundamental diagram of the present invention;
Fig. 4 is in 0.05ng/ml arrives the scope of 2.5ng/ml, and the concentration of f-PSA and nm of gold are amplified the linear relationship chart of spr signal;
Fig. 5 is in 1ng/ml arrives the 10ng/ml scope, the linear relationship chart of t-PSA concentration and spr signal.
Referring to Fig. 1, the SPR bio-sensing chip of the present invention's design is formed by 1,2,3,4,5; Wherein, 1: slide, 2: golden film, 3: mercaptan carboxylic acid, 4: ethylaminoethanol, 5:f-PSA antibody or t-PSA antibody.
Referring to Fig. 2, the detection schematic diagram of PSA SPR binary channels detection method of the present invention, wherein A is passage 1 enlarged drawing, 1: slide; 2: golden film, 3: mercaptan carboxylic acid, 4: ethylaminoethanol; 5:Anti-f-PSA, 6:f-PSA antigen, 7:Au-f-PSA antigen; B is passage 2 enlarged drawings, 1: slide, 2: golden film, 3: mercaptan carboxylic acid, 4: ethylaminoethanol, 5:Anti-t-PSA, 8:t-PSA antigen, 9: passage 1,10: passage 2.
Referring to Fig. 3, binary channels SPR pick-up unit formation of the present invention comprises 2: golden film, 9: passage 1,10: passage 2,11: laser 1; , 12: laser 2; 13: detecting device 1; 14: detecting device 2.
Embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1 plates thickness on glass slide be 40-60nm nano-gold film method, and step is following:
(1) with Piranha (H 2O 2With dense H 2SO 4Volume ratio is 1: 3 a mixed liquor) clean slide, clean up with secondary deionized water then, use N 2Dry up.
(2) slide behind the cleaning-drying being put into vacuum magnetic control spatters and carries out plated film on the sputtered films of bismuth machine:
A) plate the thick chromium film of the about 2nm of one deck earlier;
B) on the chromium film, continue the thick golden film of plating one deck 40-60nm.
Embodiment 2 particle diameters are the preparation method of the nano-Au solution of 15-40nm, and step is following:
(1) used instrument is soaked 12h with chloroazotic acid, clean dry for standby.
(2) be that 0.01% aqueous solution of chloraurate 100ml is heated to 130 ℃ with massfraction; Rapid adding massfraction is 2% trisodium citrate 2-16ml under stirring; Solution is red by colourless pulverize gradually; Become claret again, become claret continued heating 15min, heating process need stir and reflux.
(3) withdraw thermal source, continue stirring and be cooled to room temperature, change in the conical flask, preserve subsequent use under 4 ℃ of conditions.
Embodiment 3SPR chip preparation method, step is following:
(1) golden film lucifuge in 4mmol/L mercaptan carboxylic acid's alcoholic solution is soaked 12h, form one deck self assembled monolayer.
(2) with 0.1mol/L NHS and 0.4mol/L EDC mixed liquor activation mercaptan carboxylic acid terminal-COOH, solution is prepared with 10mmol/L PBS (PH7.4) damping fluid.
(3) flow injection Anti-PSA is connected on the golden film it, and the flow velocity of flow injection is 5-50 μ l/min; Zone corresponding to two passages in the SPR binary channels is fixed with f-PSA antibody and t-PSA antibody respectively;
(4) seal activation with the 1mol/L aminoethanol solution but not with the prostate antibody response-COOH, promptly make said chip.
Antigen, antibody that the present invention uses are all bought in Shanghai Linc-Bio Science Co., Ltd..
Embodiment 4 preparation Au mark f-PSA antigen methods, step is following:
(concentration is 0.63 * 10 to (1) 200 μ L nano-Au solution -9Mol/L) add 0.1mol/L K in 2CO 3Solution 10 μ L transfer pH to 9.0.
(2) add than minimum mark amount (the minimum mark amount is 2.2 μ g/ml) to the nano-Au solution that mixes up pH and Duo 50% f-PSA, normal temperature reacts 2h down.
(3) with solution at 4 ℃, centrifugal 10min under the 9000r/min condition removes the upper strata stillness of night, removes the unnecessary f-PSA that does not combine with nm of gold, adds isopyknic 10mmol/L PBS (PH7.4) damping fluid dilution, promptly makes Au mark f-PSA antigen centrifugal three times.
Embodiment 5 sample processing methods, step is following:
(1) gets venous blood on an empty stomach, pack into and contain in the vacuum tube of EDTA, leave standstill 1h under the room temperature.
(2) at 4 ℃, centrifugal 30min under the 1600r/min condition.
(3) get upper serum, remove lower floor's haemocyte.
(4) serum is refrigerated under-80 ℃ of conditions, directly inject serum during experiment and detect.
Embodiment 6 surface plasmon resonance bilateral Dow process detect f-PSA and t-PSA method simultaneously, and concrete antigen detection method principle may further comprise the steps referring to Fig. 2:
(1) described bio-sensing chip is placed in the SPR binary channels; Corresponding two sense channels of band antibody regions difference on the chip; Au is marked normal concentration f-PSA antigen, and (normal concentration f-PSA antigen gold mark process is the general labeling process; Nano-Au solution is not required,, promptly can play the effect that signal amplifies as long as contain nm of gold in the antigenic solution) flow injection through among the SPR to f-PSA detection of antibodies passage should be arranged; Normal concentration t-PSA antigen flow injection through among the SPR to t-PSA detection of antibodies passage should be arranged, and record spr signal production standard curve;
(2) with two sense channels of pretreated testing sample flow injection process SPR, and the record spr signal changes;
A) sample after centrifugal with the dilution of PBS (PH 7.4) damping fluid of isopyknic 10mmol/L;
B) flow velocity of injected sample solution is 5-50 μ l/min, is reacted to baseline and walks flat.
(3) continue to the Au mark f-PSA antigen that the passage injection embodiment 4 that f-PSA antibody should be arranged is prepared, and the record spr signal changes;
(4) according to standard working curve, confirm that the spr signal of above-mentioned different passages changes corresponding respectively f-PSA and t-PSA concentration, obtain the f/t value;
As can beappreciated from fig. 4, in the scope of 2.5ng/ml, it is linear that the concentration of f-PSA and nm of gold are amplified spr signal at 0.05ng/ml.As can be seen from Figure 5, in the 10ng/ml scope, t-PSA concentration and spr signal are linear at 1ng/ml.
In the experimentation, actual sample is detected, select 3 healthy males as the control control group, 5 PCa patients detect them respectively as experimental group, can detect f-PSA and t-PSA simultaneously with SPR bilateral Dow process of the present invention.Can find out from the experimental data of following table, compare with healthy subjects that f.t obviously reduces among the PCa patients serum.Diagnose PCa with f/t, can improve accurate rate of diagnosis, the result of the actual sample that we detect is consistent with the diagnostic result of hospital.
Figure BDA0000128893890000071

Claims (8)

1. one kind is used for the bio-sensing chip that SPR bilateral Dow process detects PSA; It is characterized in that; This chip matrix is a glass sheet, and glass sheet surface is coated with golden film, and there is the mercaptan carboxylic acid's class unimolecular layer that forms through chemical modification on golden film surface; Zone corresponding to two passages in the SPR binary channels on the unimolecular layer is fixed with f-PSA antibody and t-PSA antibody respectively, and seals remaining monomolecular avtive spot with aminoacetic acid.
2. bio-sensing chip according to claim 1 is characterized in that, described golden film thickness is 40-60nm.
3. bio-sensing chip according to claim 1 is characterized in that, described mercaptan carboxylic acid's class is that methyl unit's number is the HS (CH of 8-16 2) nCOOH.
4. bio-sensing chip according to claim 1 is characterized in that, going back spraying plating between glass sheet and the golden film has the thick chromium of one deck 2nm.
5. one kind is used for the kit that SPR bilateral Dow process detects PSA, it is characterized in that, comprises any described bio-sensing chip of claim 1-4, t-PSA antigen and Au mark f-PSA antigen.
6. kit according to claim 5 is characterized in that, described Au mark f-PSA antigen is to mix with excessive f-PSA antigenic solution, refrigerated centrifuge, obtain after removing supernatant liquor through nm of gold; Wherein said Au diameter of nano particles is 15-40nm.
7. the method that PSA detects is characterized in that, may further comprise the steps:
(1) any described bio-sensing chip of claim 1-4 is placed in the SPR binary channels; Corresponding two sense channels of band antibody regions difference on the chip mark among the normal concentration f-PSA antigen flow injection process SPR Au to f-PSA detection of antibodies passage should be arranged; Normal concentration t-PSA antigen flow injection through among the SPR to t-PSA detection of antibodies passage should be arranged, and record spr signal production standard curve;
(2) with two sense channels of pretreated testing sample flow injection process SPR, and the record spr signal changes;
(3) continue to mark f-PSA antigen, and the record spr signal changes to the passage that f-PSA antibody should be arranged is injected Au;
(4) according to standard working curve, confirm that the spr signal of above-mentioned different passages changes corresponding respectively f-PSA and t-PSA concentration, obtain the f/t value;
Described Au mark f-PSA antigen is to mix with excessive f-PSA antigenic solution, refrigerated centrifuge, obtain after removing supernatant liquor through nm of gold; Wherein said Au diameter of nano particles is 15-40nm.
8. detection method according to claim 7 is characterized in that, each normal concentration antigen, and testing sample all is 5-50 μ l/min with the flowing velocity of gold mark antigen.
CN2012100019653A 2012-01-05 2012-01-05 Biosensor chip, kit and detection method for SPR (Surface Plasmon Resonance) dual-channel method to detect specific prostate antigen Pending CN102520161A (en)

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CN104155453A (en) * 2014-07-11 2014-11-19 天津大学 Hyaluronic acid modified surface plasma resonance spectrometer chip and preparation method thereof
CN105277710A (en) * 2014-07-16 2016-01-27 林宽锯 High sensitivity LSPR (Localized Surface Plasmon Resonance) biochemical sensing kit and application method
CN107478613A (en) * 2017-08-02 2017-12-15 杭州晶百检测技术有限公司 A kind of preparation method of a variety of drug testing chips based on SPR
CN110907643A (en) * 2019-12-02 2020-03-24 中国科学院重庆绿色智能技术研究院 Preparation method of escherichia coli detection chip and detection chip
CN111273031A (en) * 2020-02-29 2020-06-12 武汉大学 Kit for biomarker detection and preparation method and application thereof
CN111351925A (en) * 2019-07-29 2020-06-30 中南大学 Paper chip capable of simultaneously detecting various aminoglycoside antibiotics, preparation and application thereof
CN114471759A (en) * 2022-01-27 2022-05-13 盖秩舶 Micro-fluidic chip based on polytetrafluoroethylene and glass and preparation method thereof

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CN103792368A (en) * 2014-01-27 2014-05-14 暨南大学 Surface plasma resonance immunosense chip as well as preparation method and application thereof
CN103792368B (en) * 2014-01-27 2015-10-07 暨南大学 A kind of surface plasma body resonant vibration immune sensing chip and preparation method thereof and application
CN104155453A (en) * 2014-07-11 2014-11-19 天津大学 Hyaluronic acid modified surface plasma resonance spectrometer chip and preparation method thereof
CN105277710A (en) * 2014-07-16 2016-01-27 林宽锯 High sensitivity LSPR (Localized Surface Plasmon Resonance) biochemical sensing kit and application method
CN107478613A (en) * 2017-08-02 2017-12-15 杭州晶百检测技术有限公司 A kind of preparation method of a variety of drug testing chips based on SPR
CN111351925A (en) * 2019-07-29 2020-06-30 中南大学 Paper chip capable of simultaneously detecting various aminoglycoside antibiotics, preparation and application thereof
CN111351925B (en) * 2019-07-29 2021-07-09 中南大学 Paper chip capable of simultaneously detecting various aminoglycoside antibiotics, preparation and application thereof
CN110907643A (en) * 2019-12-02 2020-03-24 中国科学院重庆绿色智能技术研究院 Preparation method of escherichia coli detection chip and detection chip
CN111273031A (en) * 2020-02-29 2020-06-12 武汉大学 Kit for biomarker detection and preparation method and application thereof
CN114471759A (en) * 2022-01-27 2022-05-13 盖秩舶 Micro-fluidic chip based on polytetrafluoroethylene and glass and preparation method thereof

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Application publication date: 20120627