CN104237169A - Detection method of SPR detection system based on external field modulation - Google Patents
Detection method of SPR detection system based on external field modulation Download PDFInfo
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Abstract
The invention provides a detection method of an SPR detection system based on external field modulation. The method includes the steps: 1) acquiring SPR angle scanning curves of a first sample solution and a second sample solution, calculating the sensitivity of the SPR detection system under a zero external field, and calibrating a detection dynamic range; 2) selecting a certain angle in the dynamic range as an incident angle, and carrying out external field scanning, to obtain the linear coefficient of change of the reflected light intensity along with the external field intensity; 3) introducing a to-be-measured solution, selecting an incident angle the same as that of the step 2), adjusting the applied external field intensity, allowing the detected reflected light intensity maintained in the dynamic range all the time, and reading out the external field intensity and the reflected light intensity; and 4) converting the measured reflected light intensity into an equivalent reflected light intensity under the zero external field based on the obtained linear coefficient, and then based on the sensitivity of the SPR detection system, calculating the refractive index or concentration of the to-be-measured solution by utilizing the equivalent reflected light intensity. The method can significantly expand the dynamic range of an adjustable surface plasma resonance sensor.
Description
Technical field
The present invention relates to sensor and field of sensing technologies, specifically, the present invention relates to a kind of detection method based on tunable surface plasma resonance sensor.
Background technology
Surface plasma resonance sensor is a kind of optical sensing technology that can detect metal interface near zone variations in refractive index.From the eighties in 20th century, surface plasmon resonance biosensor exempts from mark because it is outstanding, rapid sensitive and the advantage that detects in real time and obtain develop rapidly and be widely used.Be applied to protein interaction, nucleic acid interaction, food security, residues of pesticides at present, Environmental security and drug screening, the fields such as infectious disease rapid sensitive detection.
Tunable surface plasmon resonance biosensor combines based on prism and waveguide two kinds of coupling scheme the surface plasma body resonant vibration excited, concrete principle can list of references " J.Homola; Surface plasmon resonance sensors for detection of chemical and biological species; Chem.Rev., (2008) 108:462-493 ".Because tunable surface plasmon resonance biosensor is more harsh to the requirement of coupling condition, make tunable SPR reflectance curve (curve that SPR chip surface reflectivity changes with incident angle) more sharp-pointed, therefore there is higher sensitivity.At present, the detection method based on tunable surface plasmon resonance biosensor mainly contains angle scanning method and intensity detection method.Wherein, angle scanning method needs to be changed light path by physical construction, and operation easier is comparatively large, easily artificially introduces error.Intensity detection method is the angle of fixing incident light, measures the intensity of reflected light through the reflection of SPR chip surface, because the intensity of incident light is known, therefore can calculate the reflectivity of SPR chip surface, and and then obtains the characteristic such as refractive index of material to be detected.This method can be avoided being changed light path by physical construction, and simple to operate, error is relatively little.Intensity detection is generally first select the incident light of some strength, the incident angle of fixing incident light, then after SPR chip surface passes into material to be detected, measure the intensity of reflected light, calculate refractive index and other character of material to be detected according to intensity of reflected light.Its principle is as follows: the change detecting surface (referring to the SPR chip surface combining material in solution to be measured) refractive index can cause the movement of SPR reflectance curve position, Fig. 1 (a) shows the example of reflectance curve movement under an intensity detection method, because in reality detection, reflectivity corresponds to detected intensity of reflected light, in figure, replace reflectivity can not change the shape of reflectance curve with intensity of reflected light.With reference to figure 1(a), in figure, solid line is the reflectance curve of original SPR chip surface, and dotted line is the reflectance curve of the SPR chip surface combining solution to be measured.Can find out, after SPR chip surface passes into solution to be measured, SPR chip surface refractive index increases, and reflectance curve moves right.The angle fixing at certain detects reflectivity, and when surface refractive index changes, the reflectivity detected changes.Refractive index increase causes reflectance curve to move right, and under fixing incident angle, the reflectivity signals detected increases.When fixing incident angle is selected in place, and the variations in refractive index detected controls when certain scope, and the reflectivity on solution refractive index to be measured and spr sensor chip surface is changing into linear relationship, as Fig. 1 (b).Line segment in figure can represent with function Y=A+B*X, and wherein B is intensity sensitivity, and unit is RU or RIU.So just can converse material surface refractive index to be detected according to reflectivity change, thus obtain the parameters such as detection material concentration, compatibility.In quantitative analysis process, line segment in Fig. 1 (b) must keep good linear, this just requires that the moving range of the incident angle in Fig. 1 (a) and SPR reflectance curve meets some requirements: namely no matter what kind of position reflectance curve appears at, incident angle all should in the middle of its range of linearity, and the sensing range meeting above-mentioned condition is exactly the dynamic range corresponding to this incident angle.For the reflectance curve (being in fact exactly the scanning curve that reflective light intensity changes with incident angle) of original SPR chip surface, the range of linearity wherein generally between curve minimum point and peak 20% ~ 80% scope in after passing into solution to be measured, the refractive index combining the SPR chip surface of material in solution to be measured becomes large, SPR curve can be caused along X-axis (X-axis represents incident angle) direction to right translation, the range of linearity of SPR reflectance curve is also thereupon to right translation, when the refractive index of solution to be measured is excessive, this translation may cause original incident angle of light outside the range of linearity of SPR reflectance curve, namely beyond the dynamic range of the intensity detection under former angle.For tunable surface plasmon resonance biosensor, the original SPR reflectance curve of its SPR chip surface is inherently comparatively sharp-pointed, and like this, the scope of the SPR reflectance curve translation allowed is just less, cause the dynamic range of its intensity detection less, be unfavorable for actual use.
In conventional strength detection method, when detection signal is beyond dynamic detection range, need mobile SPR scanning angle, then proceed to detect.This scheme needs to be changed light path by physical construction, often affects accuracy and the consistance of experiment, and needs interrupt experiments, and rescan SPR curve and calibrate.
Therefore, the practicality of what less dynamic range was serious have impact on this New type of S PR sensor of tunable SPR, current in the urgent need to a kind of detection scheme based on tunable surface plasmon resonance biosensor had compared with great dynamic range.
Summary of the invention
The present invention proposes a kind of detection scheme based on tunable surface plasmon resonance biosensor had compared with great dynamic range.
For achieving the above object, the invention provides a kind of detection method based on tunable surface plasma resonance sensor, described tunable surface plasma resonance sensor is the SPR detection system of the tunable SPR biochip of use of a kind of outfield modulation, and described detection method comprises the following steps:
1) outfield is set to 0, refractive index or known first, second sample solution of concentration is passed into respectively in sample cell on described tunable SPR biochip, complete angle scanning respectively, obtain the SPR angle scanning curve of the first sample solution and the second sample solution; Calculate the sensitivity of 0 outer described SPR detection system after the match, and demarcate according to the range of linearity of first, second sample solution SPR angle scanning curve the dynamic range detected;
2) a certain angle in selected described dynamic range, as incident angle, is carried out outer field scan, is obtained reflective light intensity and change scanning curve with outfield strength, and then calculates the linear coefficient that reflective light intensity changes with outfield strength;
3) solution to be measured is passed in the sample cell on described tunable SPR biochip, selected and step 2) identical incident angle, when reflective light intensity exceeds over time or be about to exceed described dynamic range, keep same incident angle, regulate the outfield strength applied, make detected intensity of reflected light remain in described dynamic range, read outfield strength now and intensity of reflected light;
4) based on step 2) intensity of reflected light under current outfield strength is converted to 0 outer equivalent reflective light intensity after the match by the linear coefficient that draws, the sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this equivalent intensity of reflected light to calculate refractive index or the concentration of solution to be measured.
Wherein, in described step 1), angle scanning is carried out respectively after the match at the outer of varying strength, obtain the SPR angle scanning curve of the first sample solution and the second sample solution, demarcate the dynamic range corresponding to the outfield corresponding to each intensity according to the range of linearity of first, second sample solution SPR angle scanning curve;
In described step 3), when the reflective light intensity of solution to be measured is more than 0 during outer dynamic range after the match, by regulating outfield strength, in the dynamic range under making detection signal intensity of reflected light get back to corresponding outfield strength, and then read outfield strength and intensity of reflected light.
Wherein, the described range of linearity is the intensity of reflected light region making the linearity be greater than 0.9995.
Wherein, in described step 1), based on the range of linearity of the SPR angle scanning curve under each outfield strength, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, normalized is done to SPR angle scanning curve; Further, with the SPR angle scanning curve after the normalization of the first sample solution and the second sample solution, the normalization sensitivity of 0 outer described SPR detection system is after the match calculated;
Described step 2) in, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, the curve that the reflective light intensity that external field scan obtains changes with outfield strength does normalized;
In described step 3), according to read-out outfield strength, the normalization SPR angle scanning curve of the corresponding outfield strength drawn according to step 1), is normalized read-out intensity of reflected light;
In described step 4), based on step 2) linear coefficient of curve that changes with outfield strength of reflective light intensity after the normalization that draws, intensity of reflected light after normalization under current outfield strength is converted to 0 outer normalization equivalent reflective light intensity after the match, the normalization sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this normalization equivalent reflective light intensity meter to calculate refractive index or the concentration of solution to be measured.
Wherein, first, second sample solution described is known 1 × PBS, the 2 × PBS of refractive index or concentration, deionized water, 1:200 phosphate buffer or glycerite.
Wherein, described outfield is electric field, thermal field, magnetic field or sound field, and described outfield strength is voltage, temperature, magnetic field intensity or the sound intensity.
Compared with prior art, the present invention has following technique effect:
1, the dynamic range of tunable spr sensor is significantly expanded.
2, while expansion intensity detection method dynamic range, metrical error is suppressed.
3, while expansion intensity detection method dynamic range, higher sensitivity is kept.
Accompanying drawing explanation
The reflectance curve of SPR chip surface after Fig. 1 (a) shows original SPR chip surface and passes into solution to be measured;
Fig. 1 (b) shows the relation curve of reflectivity with solution variations in refractive index to be measured;
Fig. 2 shows the principle schematic of the SPR detection system of the outfield modulation adopted in one embodiment of the invention;
Fig. 3 shows the schematic diagram of reflectance curve for demarcating intensity sensitivity S in one embodiment of the invention;
Fig. 4 shows the schematic diagram of reflectance curve for demarcating the linear coefficient v of outfield modulation voltage and reflective light intensity in one embodiment of the invention;
Fig. 5 shows the process flow diagram based on the detection method of tunable surface plasma resonance sensor in one embodiment of the invention.
Embodiment
Below, with specific embodiment, the present invention is done by reference to the accompanying drawings and describe further.
According to first embodiment of the invention, a kind of detection method based on tunable spr sensor is provided.This detection method realizes based on a kind of SPR detection system of Electric Field Modulated.For ease of understanding, first the SPR detection system that described outfield is modulated is briefly introduced.
Fig. 2 shows the SPR detection system of the tunable spr sensor of one of the prior art-----Electric Field Modulated, and this detection system comprises: monochromatic source 1, optical module 2, high index prism 3, photo-detector 4, electric field-tunable WCSPR biochip 5, sample cell 6, microfluidic system 7, voltage source 8 and control system 9.Wherein, monochromatic source 1 and optical module 2 form directional light output unit, for providing the light beam of monochrome, linear polarization (p polarized light), collimation.Optical module 2 comprises lens combination, filter plate and the polaroid etc. of not limitting order.High index prism 3 enters electric field-tunable WCSPR biochip 5 for the light beam coupling making described directional light output unit and provide.Electric field-tunable WCSPR biochip 5 is made up of biological detection layer, upper strata metal, electrical modulation layer, lower metal and substrate from top to bottom successively.Voltage source 8 applies voltage by upper strata metal and lower metal to tunable WCSPR biochip 5, for the physical property (as refractive index, thickness) of the electrical modulation layer in tuning electric field-tunable WCSPR biochip 5.What the present embodiment adopted is carry out tuning device to electrical modulation layer refractive index, and electrical modulation layer is electrooptical modulation layer.Sample cell 6 detection material is confined to the device that electric field-tunable WCSPR biochip 5 detects surface, and its quantity, shape and size are needed to determine by detection.Photo-detector 4 is for detecting the reflection of the detection layers from electric field-tunable WCSPR biochip 5 or transmitted light intensity.Control system 9 is the software and hardware systems determined detection angles, complete outer field scan, determine to modulate outfield strength, record detection signal, analysis data process, includes but not limited to turntable controller, field generator controller, data acquisition unit, central controller and control and analysis software.Microfluidic system 7 is changed for realizing sample in sample cell, and microfluidic system 7 comprises the fluid control cleaning for biochip surface, live again, and this fluid control comprises fluid pump, sluice valve and microchannel.
The working mechanism of the SPR detection system of above-mentioned Electric Field Modulated is as follows: the light beam that monochromatic source 1 sends, through optical module 2 shaping, filtering, polarization manipulation, produces light that is monochromatic, parallel, p-type polarization and is coupled into electric field-tunable WCSPR biochip 5 by high index prism 3.Photo-detector 4 accepts the reflected light from electric field-tunable WCSPR biochip 5, is detected by the information of change to tunable WCSPR biochip 5 surface of light intensity.With the upper/lower layer metallic of electric field-tunable WCSPR biochip 5 for electrode, voltage source 8 applies electric field to electrooptical modulation layer.Electrooptical modulation layer adopts linear electrooptic material, and the pass of its variations in refractive index and extra electric field is:
Wherein d is the thickness of electrooptical modulation layer, and V is the voltage putting on electrooptical modulation layer, γ
33for the electrooptical coefficient of electrooptical modulation layer, n is the initial refractive rate of electrooptical modulation layer.As can be seen from above formula, variations in refractive index and the extra electric field of electrooptical modulation layer have linear relationship, with this ultimate principle, are modulated by the refractive index of extra electric field to electrooptical modulation layer.Microfluidic system 7 by devices such as miniflow pump, sluice valves, to injecting solution in sample cell 6 or carrying out the replacing, cleaning etc. of sample.Above-mentioned all monochromatic sources 1, photo-detector 4, microfluidic system 7, voltage source 8 etc. are all coordinated by control system 9, comprise the size of light intensity, the frequency that reflected light gathers, the position of light source, photo-detector, the flow velocity of sample solution, currency, the size of extra electric field, frequency, obtain the process etc. of data, thus obtain biochip surface information, complete the detection of bio-molecular interaction.
Below, based on the SPR detection system that above-mentioned outfield is modulated, introduce the detection method based on tunable spr sensor of the present embodiment, with reference to figure 5, the method comprises the following steps:
Step 101: when first, second sample solution utilizing refractive index known is zero to modulation electric field intensity, the intensity sensitivity S of chip demarcates.
Choose first, second sample solution that refractive index is known, respectively angle scanning is done to first, second sample solution, obtain its SPR angle scanning curve.In the present embodiment, use 1 × PBS and 2 × PBS respectively as first, second sample solution, its SPR angle scanning curve is see Fig. 3, and scan-data is as shown in table 1.
Table 1
The selection scheme of first, second sample solution above-mentioned is only exemplary, and those skilled in the art can choose arbitrarily two kinds as first, second sample solution from the known solutions such as refractive index such as 1 × PBS, 2 × PBS, deionized water, 1:200 phosphate buffer, glycerine.
With the scanning curve of 1 × PBS for benchmark, choose its linearity and be greater than the region of 0.9995 as dynamic detection range, as the angular range, theta in Fig. 3
i~ θ
j.Wherein, the concrete grammar of linearity analysis can list of references " J.Homola; Surface plasmon resonance sensors for detection of chemical and biological species; Chem.Rev.; (2008) 108:462-493 ", repeats no more herein.
Choose dynamic detection range θ
i~ θ
j1 interior θ
kas fixing detection angles, the reflective light intensity of first, second sample solution corresponding to it is respectively I
k1, I
k2.The specific refractivity of known 1 × PBS and 2 × PBS is 0.00154RIU (Refractive Index Unit, refractive index unit) simultaneously, and when can be zero to modulation electric field intensity thus, the intensity sensitivity S of chip demarcates:
So, for the detection signal I be in dynamic detection range
xits refractive index R
xcan be expressed as:
Step 102: the linear coefficient v that reflective light intensity changes with outfield modulation voltage is demarcated.
Situation when being zero for above-mentioned modulation voltage, fixed test angle is θ
ktime the sensing range of the reflection strength corresponding to dynamic detection range be I
j~ I
i, ranges of indices of refraction is R
j~ R
i.In testing process, if the reflective light intensity detecting thing has exceeded this scope, namely exceeded linear detection range, be false by the conversion relation caused in formula (2), the refractive index (i.e. detection signal strength) of the detection thing calculated is also by inaccurate.
Fig. 4 respectively illustrates when outfield is 0 and applies an intensity to chip is V
mmodulation voltage, scan the SPR reflectance curve that obtains, wherein, for the first sample solution, modulation voltage is V
mtime angle θ
kcorresponding reflected light intensity values I
k1m.
Due to outfield modulation voltage and variations in refractive index linear, namely change linear with reflective light intensity, therefore have:
I
k1m-I
k1=v(V
m-0) (3)
That is,
v=(I
k1m-I
k1)/V
m (4)
Therefore, for any outfield modulation voltage V
xunder detection signal I'
x0 corresponding outfield equivalence detection signal I
xcan be expressed as:
I
x=I'
x-vV
x (5)
Can find out, after drawing the linear coefficient v that reflective light intensity changes with outfield modulation voltage, by the I in (5) formula
xin (2) formula of substitution, under any outfield situation can be calculated, the size of determinand refractive index.
Step 103: pass into solution to be measured, when the reflective light intensity of solution to be measured has exceeded I
j~ I
iinitial dynamic linear response range time, by regulating outfield modulation voltage, make detection signal reflected light intensity values get back to I
j~ I
iscope in.Record outer field voltage V now
xwith reflected light intensity values I
x, convert according to formula (2) and (5), the refractive index of solution to be measured can be obtained.This solution avoids and regulate light path or mechanical system, ensure that the continuity of experiment and the exact correspondence of result, effectively expand the dynamic range of SPR intensity detection, substantially increase the practical value of WCSPR sensor.
Table 2 shows the linearity under initial linear region the range of linearity of SPR reflectance curve (when namely modulation voltage is 0V) of SPR reflectance curve under a SPR chip different modulating voltage and their respective refractive index sensing ranges.
Table 2
| Voltage | Sensitivity | The linearity | Refractive index sensing range |
| 0V | 25092.91 | 0.99979 | 1.3310-1.3324=0.0014 |
| -10V | 24403.88 | 0.99981 | 1.3317-1.3331=0.0014 |
| -20V | 23551.91 | 0.99982 | 1.3324-1.3338=0.0014 |
| -30V | 22438.69 | 0.99973 | 1.3331-1.3346=0.0015 |
| -40V | 21073.62 | 0.99947 | 1.3338-1.3355=0.0017 |
| -50V | 19648.24 | 0.99909 | 1.3346-1.3364=0.0018 |
| -60V | 19648.24 | 0.99757 | 1.3354-1.3372=0.0018 |
| -70V | 15969.09 | 0.99517 | 1.3362-1.3384=0.0022 |
Can find out, when modulation voltage amplitude of variation is 70V, the dynamic range that material refractive index detects is extended to 1.3310-1.3384 by 1.3310-1.3324.
Present invention also offers second embodiment, this embodiment is on the basis of previous embodiment, consider the impact that the deformation of the angle scanning curve of SPR chip after applying outfield itself causes, thus suppress the error of measurement result further, improve measuring accuracy.Still be described for the SPR detection system of the outfield modulation described in above.
In second embodiment, before described step 103, angle scanning is re-started to the SPR curve under different voltage, and reselect its range of linearity (region that the linearity is greater than 0.9995), namely the sensing range corresponding to different voltage strength is again demarcated, obtain dynamic linear region, the data obtained are as shown in table 3.
Table 3
| Voltage | Sensitivity | The linearity | Refractive index sensing range |
| 0V | 25092.91 | 0.99979 | 1.3310-1.3324=0.0014 |
| -10V | 24403.88 | 0.99981 | 1.3317-1.3331=0.0014 |
| -20V | 23551.91 | 0.99982 | 1.3324-1.3338=0.0014 |
| -30V | 22360.4 | 0.9998 | 1.3332-1.3347=0.0015 |
| -40V | 21314.11 | 0.99978 | 1.3341-1.3356=0.0015 |
| -50V | 19970.02 | 0.99974 | 1.335-1.3366=0.0016 |
| -60V | 18579.43 | 0.9998 | 1.3359-1.3376=0.0017 |
| -70V | 16995.87 | 0.99964 | 1.337-1.3389=0.0019 |
As can be seen from subordinate list 3, the linearity of curve has had obvious improvement, all more than 0.9995.For ease of operation, refractive index sensing range under different voltage can be converted to the range of linearity of the intensity of reflected light under relevant voltage.
In step 103, when the reflective light intensity of solution to be measured has exceeded I
j~ I
iinitial dynamic linear response range time, by regulating outfield modulation voltage, in the range of linearity under making detection signal reflected light intensity values get back to corresponding outfield modulation voltage, and then the reflected light intensity values measured by record, and then the refractive index of solution to be measured is calculated according to formula (2) (5).
The detection method of above-mentioned second embodiment can ensure that the SPR reflectance curve under any voltage all has the high linearity, thus while expansion intensity detection method dynamic range, suppresses metrical error.
Present invention also offers the 3rd embodiment, this embodiment, on the basis of previous embodiment, improves the sensitivity of detection further by normalized method, the detection method of this embodiment comprises the following steps:
Step 201: for the SPR chip not passing into thing to be detected, under different modulating voltage, carry out angle scanning respectively, obtains corresponding SPR reflectance curve (i.e. SPR angle scanning curve).
Step 202: do normalized to the SPR reflectance curve under each modulation voltage, obtains the normalization reduction formula of its ordinate (representing intensity of reflected light).If the ordinate value of data is y
1, y
2, y
3y
miny
max, then for the arbitrary data point ordinate y in curve Linear region
i, wherein, y
min, y
maxbe respectively minimum value and the maximal value of the ordinate in curve Linear region.Like this, normalized reduction formula can be expressed as:
203: pass into refractive index or known the first sample solution of concentration in the sample cell on tunable SPR biochip, complete angle scanning, obtain the SPR angle scanning curve of the first sample solution.
204: pass into refractive index or known the second sample solution of concentration in the sample cell on tunable SPR biochip, complete angle scanning, obtain the SPR angle scanning curve of the second sample solution.
205: the SPR angle scanning curve obtained according to step 203,204 and the refractive index of first, second known sample or concentration, calculate the sensitivity of detection system, and the size of the reflective light intensity corresponding to the range of linearity of first, second sample solution SPR curve, as its dynamic detection range.
206: the first sample solution in selected step 1), or a certain angle (angle as corresponding to the minimum point of the range of linearity) corresponding to angle scanning curve linear region of the second sample solution in step 204, carry out outer field scan, the scope that the relation curve neutral line degree selecting outfield strength and reflective light intensity to change is greater than 0.9995 is as the range of linearity of outer field scan, the ordinate (i.e. reflective light intensity) of scanning result is normalized, and then obtains the linear coefficient v that the reflective light intensity after normalization changes with outfield modulation voltage
1.
207: in tunable SPR biochip sample cell, pass into solution to be detected, scanning angle is fixed angle selected when carrying out outer field scan in step 206, carry out the detection of reflective light intensity change.When reflective light intensity is about to over time or has exceeded the reflective light intensity scope corresponding to dynamic detection range, scanning constant angle is constant, relation according to the outfield strength obtained in step 206 and dynamic detection range regulates outfield strength, make reflective light intensity get back in the corresponding range of linearity, record reflective light intensity now and modulation voltage.When data processing, first normalized is done to the ordinate of measurement result, namely according to the formula of step 202, the reflective light intensity of actual measurement is scaled the reflective light intensity after normalization, and then the linear coefficient v that the reflective light intensity after the normalization obtained according to step 206 changes with outfield modulation voltage
1, calculate the actual size of detection signal, obtain net result.
The method of the present embodiment can not only increase dynamic range, can also avoid in the modulated process of outfield because the movement of SPR reflectance curve and deformation are to the harmful effect of detection sensitivity and the linearity, thus improve tunable surface plasmon resonance biosensor institute when using outfield modulation to detect and obtain the quality of data, namely improve accuracy, minimizing error.
The sensing range that different outfields modulation voltage is corresponding after normalization and dynamic linear regional processing shown in table 4.
Table 4
| Voltage | Sensitivity | The linearity | Refractive index sensing range |
| 0V | 25782.6 | 0.99978 | 1.3310-1.3324=0.0014 |
| -10V | 25156.25 | 0.99981 | 1.3317-1.3331=0.0014 |
| -20V | 24433.37 | 0.99981 | 1.3325-1.3339=0.0014 |
| -30V | 23558.65 | 0.9998 | 1.3332-1.3347=0.0015 |
| -40V | 22676.89 | 0.99982 | 1.3341-1.3356=0.0015 |
| -50V | 21631.43 | 0.9998 | 1.335-1.3366=0.0016 |
| -60V | 20505.04 | 0.99977 | 1.336-1.3377=0.0017 |
| -70V | 19273.29 | 0.99974 | 1.337-1.3389=0.0019 |
As can be seen from Table 4, after normalized, detection sensitivity under larger modulation voltage have also been obtained larger raising, thus make under the regulation and control of outfield modulation voltage, the sensing range of refractive index without 1.3310 ~ 1.3324 during extra electric field voltage, is increased to 1.3310 ~ 1.3389 when outfield modulation voltage is 0 ~ 70V from initial.Thus when refractive index sensing range is increased to initial 5.9 times, also maintain the linearity simultaneously all the time more than 0.9997, ensure that the accuracy detecting data.
In reality test, choose the material that the refractive index such as pure water and different glucose solution is known, by the scheme of above-mentioned second embodiment, measured data reach in the effect keeping detection sensitivity and the substantially constant basis of the linearity expand sensing range.
In above-mentioned the present embodiment, SPR detection system also can use the prism of other kind, grating or other photo-couplers that described light beam coupling can be entered tunable SPR biochip to carry out alternative described high index prism 3.Photo-detector 4 can be CCD, photodiode, or other are to the sensor of light intensity sensitivity.
What deserves to be explained is, electric field-tunable WCSPR biochip 5 in above-described embodiment also can substitute with other tunable SPR biochip, as coupled plasma Wave guide resonance (Coupled Plasmon-Waveguide Resonance, referred to as CPWR), Wave-guide coupling surface plasma resonance (Waveguide-Coupled SPR, referred to as WCSPR) or other have and can carry out tuning structure by mode modulated media layer physical propertys (as refractive index, thickness) such as electricity, magnetic, heat, sound to detection signal.Particularly, described tunable dielectric layer can adopt electrooptical material, magneto-optic memory technique, thermoluminescent material, acousto-material, or other can physical property (as refractive index, thickness) changes under the effect in outfield material make.The adoptable material of described metal level comprises simple metal, alloy, metallic compound, or other can as the material of SPW carrier.Wherein, described simple metal comprises gold, silver, chromium, copper and aluminium.Described detection layers is tested substance, modifies material, label material or above-mentioned combination.Similarly, the voltage source 8 in the present embodiment also can substitute with other field generator.Field generator is various electric field, magnetic field, temperature, sound wave controllable device, and the physical property of the tunable dielectric layer of described tunable SPR biochip is undertaken tuning by field generator.
It should be noted last that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted.Although with reference to embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, modify to technical scheme of the present invention or equivalent replacement, do not depart from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (6)
1., based on a detection method for the SPR detection system of outfield modulation, comprise the following steps:
1) with refractive index or known first, second sample solution of concentration, drawn the sensitivity of 0 outer described SPR detection system after the match by angle scanning, and demarcate according to the range of linearity of first, second sample solution SPR angle scanning curve the dynamic range detected; Calculate the sensitivity of 0 outer described SPR detection system after the match, and demarcate according to the range of linearity of first, second sample solution SPR angle scanning curve the dynamic range detected;
2) a certain angle in selected described dynamic range, as incident angle, is carried out outer field scan, is obtained the linear coefficient that reflective light intensity changes with outfield strength;
3) solution to be measured is passed in the sample cell on the tunable SPR biochip of described SPR detection system, selected and step 2) identical incident angle, when reflective light intensity exceeds or be about to exceed described dynamic range, keep same incident angle, regulate the outfield strength applied, make detected intensity of reflected light remain in described dynamic range, read outfield strength now and intensity of reflected light;
4) based on step 2) intensity of reflected light under current outfield strength is converted to 0 outer equivalent reflective light intensity after the match by the linear coefficient that draws, the sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this 0 outer equivalent reflective light intensity meter after the match to calculate refractive index or the concentration of solution to be measured.
2. detection method according to claim 1, it is characterized in that, in described step 1), angle scanning is carried out respectively after the match at the outer of varying strength, obtain the SPR angle scanning curve of the first sample solution and the second sample solution, demarcate the dynamic range corresponding to the outfield corresponding to each intensity according to the range of linearity of first, second sample solution SPR angle scanning curve;
In described step 3), when the reflective light intensity of solution to be measured is more than 0 during outer dynamic range after the match, by regulating outfield strength, in the dynamic range under making detection signal intensity of reflected light get back to corresponding outfield strength, and then read outfield strength and intensity of reflected light.
3. detection method according to claim 1, is characterized in that, the described range of linearity is the intensity of reflected light region making the linearity be greater than 0.9995.
4. detection method according to claim 2, it is characterized in that, in described step 1), based on the range of linearity of the SPR angle scanning curve under each outfield strength, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, normalized is done to SPR angle scanning curve; Further, with the SPR angle scanning curve after the normalization of the first sample solution and the second sample solution, the normalization sensitivity of 0 outer described SPR detection system is after the match calculated;
Described step 2) in, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, the curve that the reflective light intensity that external field scan obtains changes with outfield strength does normalized;
In described step 3), according to read-out outfield strength, the normalization SPR angle scanning curve of the corresponding outfield strength drawn according to step 1), is normalized read-out intensity of reflected light;
In described step 4), based on step 2) linear coefficient of curve that changes with outfield strength of reflective light intensity after the normalization that draws, intensity of reflected light after normalization under current outfield strength is converted to 0 outer normalization equivalent reflective light intensity after the match, the normalization sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this normalization equivalent reflective light intensity meter to calculate refractive index or the concentration of solution to be measured.
5. detection method according to claim 1, is characterized in that, first, second sample solution described is 1 × PBS, 2 × PBS, deionized water, 1:200 phosphate buffer or glycerite.
6. detection method according to claim 1, is characterized in that, described outfield is electric field, thermal field, magnetic field or sound field, and described outfield strength is voltage, temperature, magnetic field intensity or the sound intensity.
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