CN109612953A - A kind of nanogold sensor array and its detection method and application - Google Patents
A kind of nanogold sensor array and its detection method and application Download PDFInfo
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Abstract
The present invention relates to chemical sensor fields, and in particular to a kind of nanogold sensor array and the preparation method and application thereof.Multiple sensing units can be set in the sensor, to realize the detection of unitary or polynary peptide mixed system.Based on cross-linking reaction mechanism, by the competitive reaction between peptide to be measured, nanogold and amino acid, thus the first sensing unit of building；Based on non-crosslinked reaction mechanism, with peptide to be measured complex reaction can occur for metal ion, can also neutralize the negative electrical charge of nanometer gold surface, thus the second sensing unit of building.The UV-Vis spectrum that the present invention acquires the sensor, handles spectroscopic data with N-way partial least square, realizes the quantitative analysis of multicomponent system, the advantage high with accuracy, stability is good and detection limit is low.
Description
Technical field
The present invention relates to chemical sensor fields, and in particular to a kind of nanogold sensor array and its detection method with answer
With.
Background technique
In recent decades, with the fast development of medical science, the detection of biomolecule is had received widespread attention in body fluid.
In biology and clinical medicine, have to the measurements to hundreds of peptides and protein tens of in clinical and biological sample highly sensitive
Degree, specificity, repeatability and repeatability, have increasing need for.Endogenous peptide plays an important role in the stable state of human body.It is many
Endogenous peptide is in a μm olL-1For the concentration circulating of rank in body fluid, the peptide for analyzing the concentration proposes very bio-analytical process
High requirement.Currently, common detection method has immunological method, colorimetric analysis.Chinese patent literature CN105572382A
A kind of indirect enzyme-linked immunosorbent detection method of blood pressure lowering peptide is disclosed, it, can be efficiently fast using the specific reaction of antigen and antibody
The content of TunaAI in product is detected fastly.Peptide T unaAI and protein are coupled first, immunogene is made, then will be immunized
Original annotation, which is penetrated, generates antibody, antibody purification, it is established that the standard curve of Dot-ELISA utilizes the curve meter in White Rabbit body
Calculate the content of TunaAI in sample.But often haveing the defects that cross reactivity using immune analysis, sensitivity is low, selection
Property is poor.
Colorimetric sensor is one of chemical sensor, can use the variation of color to realize the inspection of various objects
It surveys.Compared with electrochemical sensor, surface enhanced Raman technique, colorimetric sensor simple, low in cost, nothing with detection method
The advantages that needing technical professional and cumbersome operation sequence, high sensitivity, so that it be made to be highly suitable for quick trace
Amount detection.Most common nano material is nanogold (AuNPs) in colorimetric sensor, and nanogold passes through adsorption and electronegativity
Effect utilizes unitary variant A in conjunction with aptamers620/A520Realize the detection to object.There is text in the prior art
The colorimetric method of open quickly detection cysteine is offered, and is successfully applied to the measurement of cysteine content in mouse brain dialyzate,
The response measured is (9.6 ± 2.1) μm ol/L.Principle is to make point of cysteine and aspartic acid based on regulation system pH
Sub- carboxyl is negatively charged and amino is positively charged, and ion electrostatic attraction effect occurs between the two by nanogold and is connected into aggregation
State shows color and ultra-violet absorption spectrum variation and achievees the purpose that detect cysteine.Although such an approach achieves can
Depending on changing detection of the detection technique to cysteine in mouse brain, but this system detection sensitivity is to be improved, and can not examine
Survey multicomponent system.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that overcome the sensitivity of endogenous peptide detection in the prior art it is low,
Poor specificity, the defect that multicomponent system can not be detected, thus provide a kind of high sensitivity, it is easy to operate, cheap, can be simultaneously
Measure nanogold sensor array and its detection method and the application of a variety of peptides.
For this purpose, technical scheme is as follows:
A kind of nanogold sensor array, including the first sensing unit and/or the second sensing unit；
First sensing unit includes A1: nano-Au solution；And B1: amino acid solution；
Second sensing unit includes A2: the nano-Au solution of nucleotide modification；And B2: metal ion solution.
Further, in first sensing unit, the amino acid solution is the series for including at least two concentration level
Amino acid solution, it is preferred that be the series of amino acids solution of 10 concentration levels；The nano-Au solution and the series
The molar ratio of amino acid solution is 1:100-1000.
Further, the amino acid is at least one of arginine and cysteine；Preferably, the amino acid is
Arginine.
Further, the partial size of the nanogold is 5-50nm；Preferably, the partial size of the nanogold is 13nm.
Further, the nanogold is prepared by citrate reduction method.
Further, after the citrate reduction method is the following steps are included: be heated to boiling for gold salt solution, under stirring
Citrate solution is added, continues to stir, the color of reaction solution is made to become peony from yellow, then proceed to stir, stops adding
Heat is cooled to room temperature to get nano-Au solution.
Further, in second sensing unit, the metal ion solution be include that at least two concentration level is
Column metal ion solution, it is preferred that be the series metal solion of 10 concentration levels；The nucleotide modification is received
The molar ratio of rice gold solution and the series metal solion is 1:100-1000.
Further, the metal ion solution is containing following Metal Ions Cd2+、Co2+、Cr3+And Pb2+In at least one
The solution of kind；Preferably, the metal ion is Cr3+。
Further, the nucleotides sequence is classified as n dNPS composition, and the n is 15-30, and the dNPS is A, T, C or G
At least one of；Preferably, the nucleotide is one of A30, T30, C30, A21, T21, C21, T15, C15, wherein
A30:5 '-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA-3 ', T30:5 '-TTT TTT TTT TTT TTT
TTT TTT TTT TTT TTT-3 ', C30:5 '-CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC-3 ', A21:
5 '-AAA AAA AAA AAA AAA AAA AAA-3 ', T21:5 '-TTT TTT TTT TTT TTT TTT TTT-3 ', C21:
5 '-CCC CCC CCC CCC CCC CCC CCC-3 ', T15:5 '-TTT TTT TTT TTT TTT-3 ', C15:5 '-CCC
CCC CCC CCC CCC-3’。
Further, the preparation method of the nanogold of the nucleotide modification, comprising the following steps: into nano-Au solution
Nucleotide concussion reaction is added up to AuNPs-DNA compound；Wherein nanogold and the molar ratio of nucleotide are 1:10.
Further, the detection of the sensor is limited to a μm olL-1；Preferably, the detection is limited to 1.8-1.9 μ
mol·L-1.
The present invention also provides a kind of methods for detecting peptide, comprising the following steps:
First sensing unit data determination: the peptide to be measured of the known concentration of serial volume is added in nano-Au solution,
Add amino acid solution, constant volume, collecting sample wave-length coverage 230-1000nm ultraviolet-visible absorption spectroscopy, as to be measured
The original ultraviolet-visible absorption spectroscopy S of peptidesam, using pure water as reference material, at identical conditions, pure water is acquired in the wavelength
The ultraviolet-visible absorption spectroscopy of range, as reference ultraviolet-visible absorption spectroscopy Sref；
Second sensing unit data determination: the peptide to be measured of the known concentration of serial volume is added to receiving for nucleotide modification
In rice gold solution, metal ion solution, constant volume, ultravioletvisible absorption of the collecting sample in wave-length coverage 230-1000nm are added
Spectrum, the original ultraviolet-visible absorption spectroscopy S as peptide to be measuredsam, using pure water as reference material, at identical conditions, acquisition
Pure water the wave-length coverage ultraviolet-visible absorption spectroscopy, as reference ultraviolet-visible absorption spectroscopy Sref；
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
A spectrum matrix is obtained respectively, and two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, it will
Spectrum is divided into calibration set and verifying collection, and calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, verifies
Collection carries out external certificate to the Quantitative Analysis Model；
Detection: peptide to be measured is obtained into spectroscopic data, then the multidimensional offset minimum binary mould by establishing using the sensor
Type is analyzed, and predicted value is obtained.
Further, the peptide to be measured is one or both of Gly-Gly and Ala-Gln.
The present invention also provides a kind of method of above-mentioned nanogold sensor array or above-mentioned detection peptide endogenous peptide it is qualitative and
Application in terms of quantitative analysis.
Technical solution of the present invention has the advantages that
1, a kind of nanogold sensor array provided by the invention, including the first sensing unit and/or the second sensing unit；
First sensing unit includes A1: nano-Au solution；And B1: amino acid solution；Second sensing unit includes A2: core
The nano-Au solution of thuja acid modification；And B2: metal ion solution；The nanogold sensor array is single by the way that multiple sensings are arranged
Member realizes the detection of the unitary or binary mixture of endogenous peptide.The aobvious red of the nano-Au solution of dispersion, the coagulation mistake of nanogold
Solution is gradually changed to blue from red in journey, and this red change procedure to blue can be surveyed with ultraviolet-visible spectrometer,
Peptide itself to be measured plays the role of stabilizer for nano-Au solution in first sensing unit of the invention, and amino acid can benefit
Make nanogold coagulation with cross-linking reaction principle, by the competitive reaction between peptide to be measured, nanogold and amino acid, to influence nanometer
The coagulation degree of gold, thus the first sensing unit of building.In second sensing unit, due to the electrostatic repulsion between nucleic acid, nucleosides
The modified nanogold of acid is not assembled in an aqueous medium, and after adding metal ion, part metals ion is complexed with peptide to be measured
Reaction, in another part metal ion and the negative electrical charge of nanometer gold surface, so that nanogold be induced to be polymerized to.When addition metal from
There are certain relationships for the concentration of sub- one timing of concentration, the aggregation extent of nanogold and endogenous peptide, so that the second sensing of building is single
Member.
2, the method for a kind of the sensor detection peptide provided by the invention, with high sensitivity, selectivity is good, facilitates reality
Advantage.Conventional method uses univariate analysis method, and the property and concentration of determinand all can be to nanogold characteristic peak A620With
A520It has an impact, stability is poor, and sensitivity is low.It is well known that the susceptibility and detectability of sensor and the number of sensing element
Measure closely related, and the quantity of sensing element is more, and data volume to be treated is more, and the present invention utilizes UV-Vis spectrum to replace
A620/A520The ratio of value, and data are handled with N-way partial least square, the quantitative analysis of multicomponent system is realized, and mention
The high accuracy and sensitivity of analysis.
Detailed description of the invention
The fusion method of two sensing unit spectroscopic datas of Fig. 1；
10 fused uv-vis spectras of horizontal data of Fig. 2 embodiment；
The regression result of the N-PLS of Gly-Gly in Fig. 3 embodiment 1 based on N-PLS；
The regression result of the N-PLS of Gly-Gly in saliva of Fig. 4 embodiment 7 based on N-PLS model；
The regression result of the N-PLS of Gly-Gly of the Fig. 5 based on PLS model.
Specific embodiment
It is related in following embodiments:
Multidimensional offset minimum binary Fisher face principle is as follows:
Multidimensional offset minimum binary (N-PLS) is the three-dimensional matrice correction that Bro etc. is proposed based on three linear decompositions and classics PLS
Algorithm.N-PLS algorithm principle is that 3 D stereo battle array X (I × J × K) is decomposed into trilinear model:
Wherein, t is the score vector of spectrum matrix,WithIt is the light containing n spectrum (sample)
For f-th of main gene of spectrum matrix X to corresponding two load vectors, F is number of main factor, and eijk is residual error battle array.With traditional PLS phase
Together, N-PLS also decomposes concentration array y (I × 1) while decomposed spectrum battle array, and makes X and y two decomposition by iteration
Process is combined into one.The specific algorithm of N-PLS is by correcting and predicting that two parts form.
Correction portion: X (I × J × K) is calibration set spectrum battle array, and I is calibration set sample number, and J is wavelength points, and K is spectrum
The condition of measurement.
(1) X is expanded into two-dimensional matrix X0 (I × JK), and determines number of main factor F, f=1 ..., F；
(2) Z matrix is calculated
(3) singular value decomposition, [w are carried out to Z matrixK, s, wJ]=svd (Zf)
(4) it calculates For the Kronecker product (Kroneckerproduct) of matrix；
(5) t is calculatedf=Xf-1wf；
(6) it calculates
(7) u is calculatedf=yf-1qi；
(8) it calculatesWherein T f=[t1 ..., tf]；
(9) X and y, X are updatedf=Xf-1-tfwf,
(10) f=f+1 returns (3), successively finds out the F score and load of X, y.
Predicted portions: to a unknown sample spectrum battle array Xun(1 × J × K), is calculated prediction result by following steps:
(1) Xun is expanded into two-dimensional matrix Xun (1 × JK), calls the w of preservationf, bfAnd qf；
(2) it calculates
(3) it calculatesWherein Tf=[t1..., tf]
Self-service Latin partition (Bootstrap Latinpartition) principle:
Self-service Latin partition is a kind of model verification method established on the basis of cross validation and random sampling are verified, and is used
In the predictive ability and stability of classification of assessment model.In fact, it is a kind of statistical analysis side for verifying model performance
Method.The step of self-service Latin partition, is as follows:
(1) score n is suitably matched by selection, sample is divided into n group by randomly averagely or on a rough average, and
Different categories of samples number contained by ensuring every group during random partition is than consistent.
(2) cross-validation is carried out to model, selects one group of sample therein to make forecast set every time, remaining (n-1)
Group sample makees calibration set collection, by the cross validation that n times recycle, is finally used for each sample and is only used for once predicting,
And record the predicted value of each forecast set sample and corresponding true value can be according to according to the predicted value and true value of sample
Following formula calculates corresponding predicted root mean square error (the Root Mean Square Error of 1 Latin partition
ofPrediction,RMSECV)；
Here N is the quantity of sample, yi' be sample i model predication value, yiFor the actual value of sample i.
(3) during nboot times self-service, per self-service primary, the primary again random grouping arrangement of all samples progress.
(4) after nboot times self-service, model built is evaluated using the average RMSEP of forecast set sample after nboot times
Classification capacity.Calculation formula is as follows:
An available broad sense and average prediction error with this method, its main feature is that being selected in modeling process
Some variations are generated when training set and forecast set spectrum.Therefore, different data processing method pair is being evaluated and compared to this method
There is statistical significance when the influence of category of model precision.
Detection limit (LOD) principle that IUPAC is defined:
Provide that the calculation method of detection limit (LOD, limit ofdetection) is as follows according to IUPAC:
LOD=35
In above-mentioned two formula, xiWithIt is the concentration value of partial least square model prediction and the average value of sample respectively.
Nanogold the preparation method comprises the following steps: in three-necked flask be added 250mL concentration be 1mM chlorauric acid solution, in magnetic force
It is heated to boiling in blender, flow back about 5 minutes or so.One of plug is taken out, 25 millis are quickly added into its solution
The sodium citrate solution that concentration is 38.8mM is risen, plug is covered.Quickly variation occurs for the color of solution at this time, from initial light
Yellow becomes colorless and becomes black again and eventually become claret.Continue to be heated to reflux 15min or so, stop heating, stirs to molten
Liquid stops stirring again after being restored to multiple room temperature.By condensed solution by crossing film having a size of 0.45 μm of filter membrane.Obtain nanometer
The concentration of gold solution is 10nM, partial size 13nm.
The preparation method of the nanogold of nucleotide modification: taking 100 μ L concentration is the nanogold particle of 10nM, is added thereto
C15 (5 '-CCC CCC CCC CCC CCC-3 ') concussion reaction that 10 μ L concentration are 1.0 μM is up to AuNPs-DNA compound.It is real
Nucleotide used in example and comparative example is applied to be synthesized by bioengineering limited liability company.
Embodiment 1
Present embodiments provide a kind of nanogold sensor array, including the first sensing unit and the second sensing unit；
First sensing unit includes A1: nano-Au solution, concentration 10nM；
B1: arginine (Arg) solution, the series of amino acids solution including 10 concentration levels, concentration be followed successively by 1/6 μM,
2/6μM,3/6μM,4/6μM,5/6μM,6/6μM,7/6μM,8/6μM,9/6μM,10/6μM；
Second sensing unit includes A2: nanogold (AuNPs-DNA) solution of nucleotide modification, concentration 10nM；
B2:CrCl3Solution, the serial CrCl including 10 concentration levels3Solution, concentration are followed successively by 1/6 μM, 2/6 μM, 3/6
μM、4/6μM、5/6μM、6/6μM、7/6μM、8/6μM、9/6μM、10/6μM。
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
First sensing unit preparation and data determination: take respectively different volumes dipeptides 1mM Gly-Gly to be measured and
The solution of 1mMAla-Gln is added in the AuNPs solution of 100 μ L, 10nM, then 10 μ L, 20 μ L, 30 μ are added into the solution respectively
L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, Arg solution that 100 μ L concentration are 10 μM and with ultrapure water constant volume to totality
Product is 600 μ L, and the ultimate density of Gly-Gly and Ala-Gln in binary mixture (Gly-Gly/Ala-Gln) are shown in Table 1.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
Second sensing unit preparation and data determination: take respectively different volumes dipeptides 1mM Gly-Gly to be measured and
The solution of 1mMAla-Gln is added in AuNPs-DNA (C15) solution of 100 μ L, 10nM, then 10 μ are added into the solution respectively
L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, the CrCl that 100 μ L concentration are 10 μM3Solution and with ultrapure
Water constant volume is 600 μ L to total volume, and each Gly-Gly and Ala-Gln be most in binary mixture (Gly-Gly/Ala-Gln)
Final concentration is shown in Table 1.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
Obtain a spectrum matrix respectively, two spectrum matrixes permeated a spectrum matrix, data fusion mode as shown in Figure 1,
Final uv-vis spectra is obtained as shown in Fig. 2, spectrum is divided into calibration set and verifying collection, correction by self-service Latin partition
Collecting sample number is 600, and verifying collection sample number is 150；5 folding cross validations are carried out using calibration set sample uv-vis spectra to determine
The latent variable number of Gly-Gly and Ala-Gln is respectively 9 and 8, and calibration set spectrum establishes quantitative point using N-way partial least square
Model is analysed, verifying collection carries out external certificate to the Quantitative Analysis Model.
The N-PLS regression result of Gly-Gly and the N-PLS regression result of Ala-Gln are shown in Fig. 3, it can be seen from the figure that
The concentration that the prediction concentrations of Gly-Gly and Ala-Gln and N-PLS model prediction obtain is highly relevant, and all sample spots are closed
Reason is distributed in cornerwise two sides.N-PLS model can be to two kinds in Gly-Gly and Ala-Gln two end number mixing objects system
Ion provides good prediction result.When Arg and Cr3+What ten levels of the two factors of concentration were all taken into account
UV-Vis spectrum matrix carries out data fusion, then establishes N-PLS model to fused data matrix, has obtained lower friendship
Fork verifies root-mean-square error and higher related coefficient, wherein the evaluation validation-cross of the N-PLS model of quantitative analysis Gly-Gly
Root-mean-square error RMSECV=0.19 ± 0.14%, R=0.9969；The evaluation interaction of the N-PLS model of quantitative analysis Ala-Gln
Verify root-mean-square error RMSECV=1.15 ± 0.07%, R=0.9989.
It is the Gly-Gly in 50.00 μM of binary mixture for concentration for the performance of further evaluation model
It is measured under the same conditions with Ala-Gln six times, obtains six groups of spectrum.This six spectrum are carried out in advance with above-mentioned N-PLS model
It surveys, calculates detection limit with detection limit (LOD) principle that the result that prediction obtains is defined according to IUPAC.The N-PLS model of foundation
The detection limit of Gly-Gly/Ala-Gln is respectively 1.89 μM and 1.86 μM.The standard deviation (STD) of Gly-Gly and Ala-Gln point
It Wei 0.96 and 0.83.These results further demonstrate that the method for the present embodiment detects the Gly-Gly/ within the scope of a certain concentration
Ala-Gln binary mixture has good predictive ability and reproducibility.
The concentration of each component in 1 binary mixture of table (Gly-Gly/Ala-Gln)
Embodiment 2
A kind of nanogold sensor array, including the first sensing unit and the second sensing unit are present embodiments provided,
First sensing unit includes A1: nano-Au solution, concentration 10nM；
B1: cysteine solution, the series of amino acids solution including 10 concentration levels, concentration are followed successively by 1/6 μM, 2/6 μ
M,3/6μM,4/6μM,5/6μM,6/6μM,7/6μM,8/6μM,9/6μM,10/6μM；
Second sensing unit includes A2: nanogold (AuNPs-DNA (T15)) solution of nucleotide modification, concentration are
10nM；
B2:CdCl2Solution, the serial CdCl including 10 concentration levels2Solution, concentration are followed successively by 1/6 μM, 2/6 μM, 3/6
μM、4/6μM、5/6μM、6/6μM、7/6μM、8/6μM、9/6μM、10/6μM。
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
First sensing unit preparation and data determination: take respectively different volumes dipeptides 1mM Gly-Gly to be measured and
The solution of 1mMAla-Gln is added in the AuNPs solution of 100 μ L, 10nM, then 10 μ L, 20 μ L, 30 μ are added into the solution respectively
L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, cysteine solution that 100 μ L concentration are 10 μM are simultaneously arrived with ultrapure water constant volume
Total volume is 600 μ L, and the ultimate density of Gly-Gly and Ala-Gln in binary mixture (Gly-Gly/Ala-Gln) are shown in Table
1。
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
Second sensing unit preparation and data determination: take respectively different volumes dipeptides 1mM Gly-Gly to be measured and
The solution of 1mMAla-Gln is added in AuNPs-DNA (T15) solution of 100 μ L, 10nM, then 10 μ are added into the solution respectively
L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, the CdCl that 100 μ L concentration are 10 μM2Solution and with ultrapure
Water constant volume is 600 μ L to total volume, and each Gly-Gly and Ala-Gln be most in binary mixture (Gly-Gly/Ala-Gln)
Final concentration is shown in Table 1.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
A spectrum matrix is obtained respectively, and two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, it will
Spectrum is divided into calibration set and verifying collection, and calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, verifies
Collection carries out external certificate to the Quantitative Analysis Model；
Spectroscopic data of the peptide to be measured in the sensor is acquired, then the multidimensional partial least square model by establishing carries out
Analysis, obtains predicted value.
Embodiment 3
A kind of nanogold sensor array, including the first sensing unit and the second sensing unit are present embodiments provided,
First sensing unit includes A1: nano-Au solution, and concentration is 10nM (nM nmol/L)；
B1:Arg solution, the series of amino acids solution including 1 concentration level, concentration be 1 μM (μM be mol/L)；
Second sensing unit includes A2: nanogold (AuNPs-DNA (C30)) solution of nucleotide modification, concentration are
10nM；
B2:CrCl3Solution, the serial CrCl including 1 concentration level3Solution, concentration are 5/6 μM.
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
The preparation and Data Detection of first sensing unit: the peptide 1mM Gly-Gly solution to be measured of different volumes is taken to add respectively
In the AuNPs solution for entering 100 μ L, 10nM, then Arg solution that 60 μ L concentration are 10 μM is added into the solution respectively and with ultrapure
Water constant volume is respectively 14,30,50,64,80 μM for the ultimate density of 600 μ L, Gly-Gly to total volume.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Gly-Gly to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
The preparation and Data Detection of second sensing unit: the solution of the peptide 1mM Gly-Gly to be measured of different volumes is taken respectively
It is added in AuNPs-DNA (C30) solution of 100 μ L, 10nM, then it is 10 μM that 50 μ L concentration are added into the solution respectively
CrCl3Solution and with ultrapure water constant volume to total volume be 600 μ L, Gly-Gly ultimate density be respectively 14,30,50,64,80 μ
M。
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Gly-Gly to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
A spectrum matrix is obtained respectively, and two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, it will
Spectrum is divided into calibration set and verifying collection, and calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, verifies
Collection carries out external certificate to the Quantitative Analysis Model；
Spectroscopic data of the peptide to be measured in the sensor is acquired, then the multidimensional partial least square model by establishing carries out
Analysis, obtains predicted value.
Embodiment 4
A kind of nanogold sensor array, including the first sensing unit and the second sensing unit are present embodiments provided,
First sensing unit includes A1: nano-Au solution, and concentration is 10nM (nM nmol/L)；
B1:Arg solution, the series of amino acids solution including 1 concentration level, concentration be 4/6 μM (μM be mol/L)；
Second sensing unit includes A2: nanogold (AuNPs-DNA (C15)) solution of nucleotide modification, concentration are
10nM；
B2:CrCl3Solution, the serial CrCl including 1 concentration level3Solution, concentration are 5/6 μM.
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
The preparation and Data Detection of first sensing unit: the peptide 1mMAla-Gln solution to be measured of different volumes is taken to be added respectively
In the AuNPs solution of 100 μ L, 10nM, then be added respectively into the solution 40 μ L concentration be 10 μM Arg solution and use ultrapure water
Constant volume is respectively 18,40,54,66,92 μM for the ultimate density of 600 μ L, Ala-Gln to total volume.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Ala-Gln to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
The preparation and Data Detection of second sensing unit: the solution of the peptide 1mMAla-Gln to be measured of different volumes is taken to add respectively
Enter in AuNPs-DNA (C15) solution of 100 μ L, 10nM, then the CrCl that 50 μ L concentration are 10 μM is added into the solution respectively3
Solution and with ultrapure water constant volume to total volume be 600 μ L, Ala-Gln ultimate density be respectively 18,40,54,66,92 μM.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Ala-Gln to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
A spectrum matrix is obtained respectively, and two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, it will
Spectrum is divided into calibration set and verifying collection, and calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, verifies
Collection carries out external certificate to the Quantitative Analysis Model；
Spectroscopic data of the peptide to be measured in the sensor is acquired, then the multidimensional partial least square model by establishing carries out
Analysis, obtains predicted value.
Embodiment 5
A kind of nanogold sensor array, including the first sensing unit are present embodiments provided,
First sensing unit includes: A1, nano-Au solution, concentration 10nM；
B1, arginine solution, the series of amino acids solution including 10 concentration levels, concentration be followed successively by 1/6 μM, 2/6 μM,
3/6μM,4/6μM,5/6μM,6/6μM,7/6μM,8/6μM,9/6μM,10/6μM；
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
The preparation of first sensing unit and data determination: the peptide 1mM Gly-Gly solution to be measured of different volumes is taken to be added respectively
In the AuNPs solution of 100 μ L, 10nM, then 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ are added into the solution respectively
L, 80 μ L, 90 μ L, arginine solution that 100 μ L concentration are 10 μM and be 600 μ L, Gly-Gly with ultrapure water constant volume to total volume
Ultimate density be 14,30,50,64,80 μM.Each concentration of Gly-Gly includes the sample of ten arginine concentrations gradients, example
Such as, Gly-Gly concentration, which is 80 μM, ten samples, and in ten samples arginic concentration is respectively 1/6 μM, 2/6 μM, 3/6 μ
M、4/6μM、5/6μM、6/6μM、7/6μM、8/6μM、9/6μM、10/6μM。
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Gly-Gly to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, according to sensing unit
Spectrum is divided into calibration set and verifying collects by the uv-vis spectra of acquisition, and calibration set spectrum uses N-way partial least square
Quantitative Analysis Model is established, verifying collection carries out external certificate to the Quantitative Analysis Model；
Spectroscopic data of the peptide to be measured in the sensor is acquired, then the multidimensional partial least square model by establishing carries out
Analysis, obtains predicted value.
Embodiment 6
A kind of nanogold sensor array, including the second sensing unit are present embodiments provided,
Second sensing unit includes: nanogold (AuNPs-DNA (T15)) solution of A2, nucleotide modification, and concentration is
10nM；
B2、CrCl3Solution, the serial CrCl including 10 concentration levels3Solution, concentration are followed successively by 1/6 μM, 2/6 μM, 3/6
μM、4/6μM、5/6μM、6/6μM、7/6μM、8/6μM、9/6μM、10/6μM。
The present embodiment additionally provides a kind of method using above-mentioned sensor array detection peptide, comprising the following steps:
The preparation of second sensing unit and data determination: the solution of the peptide 1mMAla-Gln to be measured of different volumes is taken to be added respectively
In AuNPs-DNA (C15) solution of 100 μ L, 10nM, then 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ are added into the solution respectively
L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, the CrCl that 100 μ L concentration are 10 μM3Solution and with ultrapure water constant volume to total volume be 600 μ
The ultimate density of L, Ala-Gln are 18,40,54,66,92 μM.Each concentration of Ala-Gln includes ten CrCl3Concentration gradient
Sample, for example, Ala-Gln concentration, which is 92 μM, ten samples, a CrCl in ten samples3Concentration be respectively 1/6 μM, 2/
6μM、3/6μM、4/6μM、5/6μM、6/6μM、7/6μM、8/6μM、9/6μM、10/6μM。
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as peptide Ala-Gln to be measured；Using pure water as reference material, in identical item
Under part, acquire ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm, step-length 1nm, as with reference to it is ultraviolet can
See absorption spectrum Sref.
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units
A spectrum matrix is obtained respectively, and two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, it will
Spectrum is divided into calibration set and verifying collection, and calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, verifies
Collection carries out external certificate to the Quantitative Analysis Model；
Spectroscopic data of the peptide to be measured in the sensor is acquired, then the multidimensional partial least square model by establishing carries out
Analysis, obtains predicted value.
Embodiment 7
Conduct after quantitative Gly-Gly and Ala-Gln is added into saliva for nanogold sensor array based on embodiment 1
Sample to be tested, to binary system in saliva sample (Gly-Gly/Ala-Gln) quantitative analysis.The N- of Gly-Gly in saliva sample
The N-PLS regression result of Ala-Gln is shown in Fig. 4 in PLS regression result and saliva sample.Although there is more interference in saliva sample
Factor, but the actual concentration of Gly-Gly and Ala-Gln is the same as the result of the N-PLS model prediction based on nanogold sensor array
Height meets.
Comparative example 1
Nanogold, the preparation method is the same as that of Example 1 for the nanogold of nucleotide modification；
Take the solution of dipeptides the 1mM Gly-Gly and 1mMAla-Gln to be measured of different volumes that 100 μ L are added respectively, 10nM's
In AuNPs solution, then 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L are added into the solution respectively,
Arg solution that 100 μ L concentration are 10 μM and be 600 μ L, binary mixture (Gly-Gly/ with ultrapure water constant volume to total volume
Ala-Gln the ultimate density of Gly-Gly and Ala-Gln in) are shown in Table 1.
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
Take the solution of dipeptides the 1mM Gly-Gly and 1mMAla-Gln to be measured of different volumes that 100 μ L are added respectively, 10nM's
In AuNPs-DNA (C15) solution, then 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L are added into the solution respectively,
80 μ L, 90 μ L, the CrCl that 100 μ L concentration are 10 μM3Solution and with ultrapure water constant volume to total volume be 600 μ L, two end number mixing body
The ultimate density of each Gly-Gly and Ala-Gln are shown in Table 1 in system (Gly-Gly/Ala-Gln).
30min is placed, acquires above-mentioned mixed solution in the ultraviolet-visible absorption spectroscopy of wave-length coverage 230-1000nm, step-length
Original ultraviolet-visible absorption spectroscopy Ssam for 1nm, as dipeptides Gly-Gly and Ala-Gln to be measured；Using pure water as reference substance
Matter acquires ultraviolet-visible absorption spectroscopy of the pure water in wave-length coverage 230-1000nm at identical conditions, and step-length 1nm makees
To refer to ultraviolet-visible absorption spectroscopy Sref.
: S=Ssam-Sref is calculated according to following formula to use the spectroscopic data of the Arg of various concentration and Cr3+
Partial Least Squares (PLS) establishes Quantitative Analysis Model, and the relevant parameter of corresponding model is shown in Table 2.
As shown in table 2, Gly-Gly and Ala-Gln, different Arg are detected in binary mixture (Gly-Gly/Ala-Gln)
And Cr3+The result of the corresponding PLS model of concentration.For quantitative analysis Gly-Gly, work as VArg=60 μ L, VCr 3+R reaches when=50 μ L
To maximum value, RMSECV reaches minimum value.For quantitative analysis Gly-Gly, work as VArg=40 μ L, VCr 3+R reaches when=100 μ L
Maximum value, RMSECV reach minimum value.Therefore, by VArg=60 μ L, VCr 3+=50 μ L are applied to quantitative analysis Gly-Gly (figure
5a), it is 11.17 ± 0.16 μM that the R of the model, which reaches 0.8798, RMSECV,.By VArg=40 μ L, VCr 3+=100 μ L are applied to fixed
Amount analysis Ala-Gln (Fig. 5 b), it is 5.60 ± 0.18 μM that the R of the model, which reaches 0.9742, RMSECV,.In lower difference of most having ready conditions
The PLS Quantitative Analysis Model of foundation, the R of two components is lower, RMSECV higher, and this method is unsatisfactory for wanting for accurate quantitative analysis
It asks.And a certain condition cannot be found to make the Quantitative Analysis Model of two components while being optimal.
Table 2 is based on different Arg and Cr3+The parameter of the PLS model of concentration binary mixture
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (11)
1. a kind of nanogold sensor array, which is characterized in that including the first sensing unit and/or the second sensing unit；
First sensing unit includes A1: nano-Au solution；And B1: amino acid solution；
Second sensing unit includes A2: the nano-Au solution of nucleotide modification；And B2: metal ion solution.
2. sensor array according to claim 1, which is characterized in that in first sensing unit, the amino acid
Solution is the series of amino acids solution for including at least two concentration level, it is preferred that for the serial amino of 10 concentration levels
Acid solution；The molar ratio of the nano-Au solution and the series of amino acids solution is 1:100-1000.
3. sensor array according to claim 1 or 2, which is characterized in that the amino acid is arginine and half Guang ammonia
At least one of acid；Preferably, the amino acid is arginine.
4. sensor array according to claim 1 or 2, which is characterized in that the partial size of the nanogold is 5-50nm；It is excellent
Choosing, the partial size of the nanogold is 13nm.
5. sensor array according to claim 1, which is characterized in that in second sensing unit, the metal from
Sub- solution is the series metal solion for including at least two concentration level, it is preferred that is the series of 10 concentration levels
Metal ion solution；The nano-Au solution of the nucleotide modification and the molar ratio of the series metal solion are 1:100-
1000。
6. sensor array according to claim 1 or 5, which is characterized in that the metal ion solution is containing as follows
Metal Ions Cd2+、Co2+、Cr3+And Pb2+At least one of solution；Preferably, the metal ion is Cr3+。
7. sensor array described according to claim 1 or 2 or 5, which is characterized in that the nucleotides sequence is classified as n dNPS
Composition, the n are 15-30, and the dNPS is at least one of A, T, C or G；Preferably, the nucleotide be A30, T30,
One of C30, A21, T21, C21, T15, C15, wherein A30:5 '-AAA AAA AAA AAA AAA AAA AAA AAA
AAA AAA-3 ', T30:5 '-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3 ', C30:5 '-CCC CCC
CCC CCC CCC CCC CCC CCC CCC CCC-3 ', A21:5 '-AAA AAA AAA AAA AAA AAA AAA-3 ', T21:
5 '-TTT TTT TTT TTT TTT TTT TTT-3 ', C21:5 '-CCC CCC CCC CCC CCC CCC CCC-3 ', T15:
5 '-TTT TTT TTT TTT TTT-3 ', C15:5 '-CCC CCC CCC CCC CCC-3 '.
8. sensor array described according to claim 1 or 2 or 5, which is characterized in that the detection of the sensor is limited to μ
mol·L-1；Preferably, the detection is limited to 1.8-1.9 μm of olL-1。
9. a kind of method using claim 1-8 described in any item sensor arrays detection peptides, which is characterized in that including with
Lower step:
First sensing unit data determination: the peptide to be measured of the known concentration of serial volume is added in nano-Au solution, then plus
Enter amino acid solution, constant volume, collecting sample wave-length coverage 230-1000nm ultraviolet-visible absorption spectroscopy, as peptide to be measured
Original ultraviolet-visible absorption spectroscopy Ssam, using pure water as reference material, at identical conditions, pure water is acquired in the wave-length coverage
Ultraviolet-visible absorption spectroscopy, as reference ultraviolet-visible absorption spectroscopy Sref；
Second sensing unit data determination: the peptide to be measured of the known concentration of serial volume is added to the nanogold of nucleotide modification
In solution, metal ion solution, constant volume, ultravioletvisible absorption light of the collecting sample in wave-length coverage 230-1000nm are added
Spectrum, the original ultraviolet-visible absorption spectroscopy S as peptide to be measuredsam, using pure water as reference material, at identical conditions, acquire pure
Water the wave-length coverage ultraviolet-visible absorption spectroscopy, as reference ultraviolet-visible absorption spectroscopy Sref；
It establishes multidimensional partial least square model: being calculated according to following formula: S=Ssam-Sref, two sensing units obtain respectively
A spectrum matrix, two spectrum matrixes are permeated a spectrum matrix, final uv-vis spectra is obtained, spectrum is drawn
It is divided into calibration set and verifying collection, calibration set spectrum establishes Quantitative Analysis Model using N-way partial least square, and verifying collection is to institute
It states Quantitative Analysis Model and carries out external certificate；
Detection: acquiring spectroscopic data of the peptide to be measured in the sensor, then the multidimensional partial least square model by establishing into
Row analysis, obtains predicted value.
10. the method for detection peptide according to claim 9, which is characterized in that the peptide to be measured is Gly-Gly and Ala-
One or both of Gln.
11. sensor array according to claim 1-8 or the described in any item detection peptides of claim 9-10
Application of the method in terms of endogenous peptide qualitative and quantitative analysis.
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