CN105319164B - A kind of biochip and its manufacturing method and detection method for OIRD detection method - Google Patents
A kind of biochip and its manufacturing method and detection method for OIRD detection method Download PDFInfo
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Abstract
The present invention provides a kind of biochip for oblique incident ray reflection difference detection method, comprising: substrate is made of silicon or germanium；Fixed to the biological sample on the substrate.Can also have transparent buffer layer between the substrate and the biological sample.The present invention also provides the detection method of this biochip and manufacturing methods.
Description
Technical field
The present invention relates to a kind of biochip and its detection methods, more particularly to one kind to be used for oblique incident ray difference in reflection
The biochip of (oblique-incidence reflectivity difference, OIRD) detection method and its manufacturer
Method and detection method.
Background technique
Life science is a complicated system, as albumen, nucleic acid, the every one kind of carbohydrate all contain up to ten thousand kinds it is different
Biomolecule, small molecule are even more countless.Therefore the detection and its repercussion study of biomolecule are in life science
Basic research, medical research, newtype drug research and development and the fields such as clinic be all it is particularly significant and significant, have evolved into
For one of Disciplinary Frontiers the most active in life science.
In order to study the characteristic and intermolecular interaction of numerous biomolecule, 20th century the nineties there is life
Object chip technology, biochip is once parallelizable while detecting the interaction of a biological sample points up to ten thousand, is referred to as 20
One of the trace analysis having an epoch-marking significance in century.But up to the present, in terms of detecting biochip, or with glimmering
Light is that the labeling method of representative dominates.Not only process is complicated, time-consuming and laborious at high cost for labelling method, especially mark molecule
Introducing, may influence or change be detected biomolecule structure and activity.Therefore life science is badly in need of unmarked, high
The detection technique and method of flux.
Developed OIRD detection method in recent years, can it is unmarked, with high throughput detection and research biomolecule and its mutually
Effect.The basic principle of OIRD detection method is to keep a branch of periodically variable modulation light between p-polarization and s polarization oblique
It is mapped to tested substance (such as biological sample on biochip) surface, and measure two polarized components of s and p of reflected light
Difference, thus variation and characteristic of the detection and research material surface on micro/nano-scale.Fig. 1 shows an OIRD detection device
Basic schematic diagram.As shown in Figure 1, OIRD detection device specifically includes that He-Ne laser 11, for exporting p-polarization light；Light
Modulator 12 is played, it is 50 kHz that the p-polarization light for He-Ne laser 11 to be exported, which is modulated to frequency (Ω), in p-polarization and s
Periodically variable modulation light between polarization；Phase-shifter 13 can introduce one between the p-polarization and s polarized component of modulation light
Fixed phase difference is adjusted for fundamental frequency signal；First lens 14, it is oblique to be mapped to detected biology after focusing modulation light
The surface of sample 16；Lens 18, for focusing by the reflected light of 16 surface reflection of biological sample；Analyzer 19, by adjusting it
Angle between optical axis and direction of polarized light, is adjusted frequency-doubled signal；Photodiode detector 120, for detecting warp
The reflected light of polarization analysis device 19 is crossed, and converts light signals into electric signal；First, second lock-in amplifier 121,122, respectively
The fundamental frequency and frequency-doubled signal of reflected light difference are detected, the data generated are by computer acquisition and processing.For different biologies
Interaction between sample or the various concentration or biomolecule of same sample, fundamental frequency that OIRD detection device obtains and times
Frequency signal will be different, to can get the information to interact between biomolecule or biomolecule.
About the further detail of OIRD detection biochip method, such as it can refer to document 1:Xu Wang, et
Al., J Appl Phys, 2010,107,063109, document 2:S.Liu et al., Appl.Phys.Lett., 2004,104,
163701, document 3:He Liping, et al.Sci China Phys Mech Astron, 2014,57,615, Chinese invention
Patent ZL201010128589.5, ZL200810057538.4, ZL200810101699.5 etc..
Existing result of study show OIRD detection method be at present it is unmarked, with high throughput detection biochip most
One of good method.But it is still necessary to further increase for the detection sensitivity and resolution ratio of current OIRD detection method.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of biochips for OIRD detection method, and detection spirit can be improved
Sensitivity and resolution ratio.
The present invention provides a kind of biochips for oblique incident ray reflection difference detection method, comprising:
Substrate is made of silicon or germanium；
Fixed to the biological sample on the substrate.
The biochip provided according to the present invention, further includes the container to form fluid cavity, which has transparent window, institute
It states biological sample to be located in the fluid cavity, and the biological sample is exposed to oblique incident ray difference in reflection by the transparent window
Light is detected used in detection method.
The biochip provided according to the present invention, the biological sample are fixed on the substrate by active layer.
The biochip provided according to the present invention, wherein also having transparent caching between the substrate and the biological sample
Layer, selects the thickness of the buffer layer, so that in oblique incident ray reflection difference detection method, can:
I) fundamental frequency signal and frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method are obtained simultaneously；Or
Ii the intensity of the fundamental frequency signal in the signal of the oblique incident ray reflection difference detection method) is made to be equal to its negative maximum
Or its positive maximum；Or
Iii the intensity of the frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method) is made to be equal to its negative maximum
Value or its positive maximum.
The biochip provided according to the present invention, wherein the thickness of the buffer layer is selected as making the fundamental frequency signal
Intensity is more than the 20% of its negative maximum or its positive maximum, at the same make the frequency-doubled signal intensity be more than its negative maximum or
The 20% of its positive maximum.
The biochip provided according to the present invention, wherein the material of the transparent buffer layer is SiO2、SrTiO3 LaAlO3
Or polymer.
The biochip provided according to the present invention, wherein the biological sample is fixed to the buffer layer by active layer
On.
The biochip provided according to the present invention, wherein the buffer layer is by the activation material suitable for the fixed biological sample
Material is constituted, and the biological sample is on the buffer layer.
The oblique incident ray that the present invention also provides a kind of for detecting biochip reflects difference detection method, comprising:
A predetermined value is set by the value of back end signal, which makes the signal of oblique incident ray reflection difference detection method
Intensity be at least up to the 60% of the maximum intensity that the signal strength can reach；
The detection of oblique incidence light reflection difference method is carried out to the biochip.
The present invention also provides a kind of methods for manufacturing biochip, comprising:
The substrate being made of silicon or germanium is provided；
Immobilizing biological samples on substrate.
The present invention also provides a kind of methods for manufacturing biochip, comprising:
The substrate being made of silicon or germanium is provided；
Transparent buffer layer is formed on the substrate；
The immobilizing biological samples on the transparent buffer layer,
The thickness of the buffer layer is selected, so that in oblique incident ray reflection difference detection method, can:
I) fundamental frequency signal in the signal of the oblique incident ray reflection difference detection method and the frequency multiplication are obtained simultaneously
Signal；Or
Ii the intensity of the fundamental frequency signal in the signal of the oblique incident ray reflection difference detection method) is made to be equal to its negative maximum
Or its positive maximum；Or
Iii the intensity of the frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method) is made to be equal to its negative maximum
Value or its positive maximum.
To sum up, the present invention provides a kind of biochip based on silicon substrate or germanium substrate is greatly improved OIRD detection
The sensitivity and resolution ratio of method.
Further, according to an aspect of the present invention, a kind of biochip for being based on " four layer models " is additionally provided,
One buffer layer is set between silicon or germanium substrate and biological sample.By reasonably selecting the thickness of buffer layer, the base of OIRD can be made
Frequently/or frequency-doubled signal reach maximum value, or OIRD can be made to detect while obtaining fundamental frequency and frequency multiplication two paths of signals, and believe two-way
Number intensity it is larger, to improve the detection sensitivity and resolution ratio of OIRD detection method, and can be fitted according to two paths of signals
It is detected the certain physical characteristics of biological sample out.
In addition, the OIRD detection method provided by the invention without back end zeroing, can further improve the spirit of OIRD signal
Sensitivity and resolution ratio.
Detailed description of the invention
Embodiments of the present invention is further illustrated referring to the drawings, in which:
Fig. 1 is the structural schematic diagram of OIRD detection device in the prior art；
Fig. 2 is the structural schematic diagram of biochip used in OIRD detection method in the prior art；
Fig. 3 is the structural schematic diagram according to the biochip based on " three layer model " of one embodiment of the present of invention；
Fig. 4 is the structural schematic diagram of the biochip based on " four layer models " of another embodiment according to the present invention；
Fig. 5 a and Figure 5b shows that in biochip according to an embodiment of the invention when carrying out OIRD detection
Light path schematic diagram；
Fig. 6 a and Fig. 6 b show OIRD fundamental frequency according to an embodiment of the invention and frequency-doubled signal intensity with buffering
The relationship of layer thickness variation；
Fig. 7 a and Fig. 7 b are according to one embodiment of present invention and the OIRD signal two-dimensional scanning figure of comparative example；
Fig. 8 a and Fig. 8 b show OIRD fundamental frequency according to still another embodiment of the invention and frequency-doubled signal intensity with slow
Rush the relationship of layer thickness variation；
Fig. 9 a and Fig. 9 b show OIRD fundamental frequency according to another embodiment of the invention and frequency-doubled signal intensity with slow
Rush the relationship of layer thickness variation.
Specific embodiment
Up to the present, there are no the dedicated biochips of OIRD detection method, therefore OIRD detection method uses at present
Biochip be all made of the structure of biochip used in fluorescent marker method etc..Fig. 2 shows the current detection sides OIRD
The structure of biochip used in method, comprising: glass substrate 17 (is usually made of glass slide)；It is attached to glass substrate
Active layer 161 on 17；Multiple biological samples 16, are fixed on active layer 161 in the form of rectangular, monolayer；Stream
Body cavity 15 is covered in glass substrate 17, if in fluid cavity 15 being air/or buffering liquid, can directly detect biochip
The biological sample on surface.If the liquid of fluid cavity is reaction solution, the anti-of biological sample and reaction solution on biochip can detect
Answer dynamic process and its reaction result.
Since 17 surface of glass substrate is generally poor to the adhesion of biological sample 16, usually require in glass substrate
One layer of active layer 161 is set on 17 surfaces, to combine biological sample 16.In general, the very thin thickness of active layer 161, therefore
When detecting biological sample with OIRD detection method, influence of the active layer 161 to testing result can be ignored.Therefore conventional OIRD
In detection method, glass substrate 17, biological sample 16 and fluid cavity 15 this three layers effect in biochip is only considered, so
It is the biochip based on " three layer model " that this biochip, which is also referred to as,.
When detecting, it is generated using OIRD detection device (such as OIRD detection device as shown in Figure 1) a branch of in p-polarization
S polarization between cyclically-varying and between p-polarization component and s polarized component with certain phase difference modulation light L, and
Modulation light L focusing is retreaded and is incident on biochip, the surface of biological sample 16 is incident on after glass substrate 17, is reflected
Light is by the polarization analysis device 19 in OIRD detection device (such as OIRD detection device as shown in Figure 1), then by photoelectricity two
Pole pipe detector 120 receives, and the signal that photodiode detector 120 exports is put through the first, second lock-in amplifier 121,122
Greatly, OIRD signal is finally obtained.
It can be seen that being all made of transparent substrates (example in this biochip based on " three layer model " in the prior art
Such as glass substrate).When carrying out OIRD detection, the modulation light L for detection is incident on the transparent substrates side of biochip simultaneously
Reach the surface of biological sample.Then, a part of light in incident modulation light L is reflected, another part (and it is usually larger
Part) light can be transmitted through biological sample and fluid cavity 15, becomes without using up (the part light is not shown in Fig. 2).
According to an aspect of the invention, there is provided a kind of biochip substitutes existing life using silicon or germanium as substrate
Glass substrate in object chip is greatly improved the intensity of OIRD signal.In addition, silicon and germanium both materials (including monocrystalline and more
It is brilliant) after precise polished, it can be easily attained the roughness of atomic scale, greatly reduction interface roughness is to optical signal
It influences, improves resolution ratio.And the roughness of this atomic scale is that glass substrate institute is unapproachable.Therefore, using silicon or germanium
As the substrate of biochip, the intensity of OIRD signal can be improved, reduce the influence of interface roughness, to improve OIRD spy
The sensitivity and resolution ratio of survey.
The structure of the biochip of one aspect is as shown in Figure 3 according to the present invention, comprising: opaque substrate 32, by silicon or
Germanium is constituted；The active layer 33 being attached on substrate 32；Multiple biological samples 31, are fixed on active layer 33.The biochip
It is fixed in the fluid cavity 34 of a container 37 formation.Wherein the container 37 has a transparent window 36, the biology of biochip
Sample 31 and the transparent window 36 are staggered relatively, so that biological sample 31 be made to be exposed to detection light L by the transparent window 36.
Therefore, which is used for sealed fluid chamber 34, on the other hand can pass through detection light L.It can be mentioned in fluid cavity 34
For reaction solution, for being reacted with biological sample 31.
The very thin thickness of active layer 33, usually about 1nm, therefore its influence to testing result can be ignored.Therefore, Fig. 3
Shown in biochip be the above biochip based on " three layer model ".
According to another aspect of the present invention, provide it is a kind of can further improve detection sensitivity and resolution ratio based on
The biochip of " four layer models ".Present applicant is draped over one's shoulders in the Chinese patent application application No. is 2014102229528
A kind of biochip for being based on " four layer models " is revealed, by setting buffer layer, the detection sensitivity of OIRD detection method can be improved
And resolution ratio.But the biochip uses transparent glass substrate, rather than silicon substrate or germanium substrate in the application.
The knot of the biochip based on " four layer models " according to a further aspect of the invention is introduced below in conjunction with attached drawing
Structure and its advantage.
The structure of the biochip based on " four layer models " according to a further aspect of the invention is as shown in Figure 4, comprising: impermeable
Bright substrate 42, is made of silicon or germanium；Transparent buffer layer 45 on substrate 42；The active layer 43 being attached on transparent buffer layer 45；
Multiple biological samples 41, are fixed on active layer 43.The biochip is fixed in the fluid cavity 44 of a container 47 formation.
Wherein the container 47 has a transparent window 46, and the biological sample 41 of biochip and the transparent window 46 are staggered relatively, thus
Biological sample 41 is set to be exposed to detection light L by the transparent window 46.Therefore, 46 one side of transparent window is for sealing stream
On the other hand body cavity 44 can pass through detection light L.It can provide reaction solution in fluid cavity 44, for reacting with biological sample 41.
The very thin thickness of active layer 43, usually about 1nm, therefore its influence to testing result can be ignored.Therefore, Fig. 4
Shown in biochip, only need to consider this four layers effect of substrate 42, buffer layer 45, biological sample 41 and fluid cavity 44,
So biochip shown in Fig. 4 is referred to as the biochip for being based on " four layer models ".
By theoretical calculation it is found that the obtained OIRD of this biochip based on " four layer models " in the present invention believes
In number, all variations of generating period with the change of the thickness of buffer layer 45 of the intensity of fundamental frequency and frequency-doubled signal.Hereinafter
Theoretical calculation and derivation process based on four layer models will be provided.
Fig. 5 a and Figure 5b shows that when carrying out OIRD detection, the light path schematic diagram in four layer model biochips is (wherein
Active layer 43 is omitted due to very thin).Wherein Fig. 5 a is the region (packet that biochip surface is not bound with biological sample 41
Include 44 3 layers of substrate 42, buffer layer 45 and fluid cavity) light path schematic diagram, and Fig. 5 b be biochip surface combine biological sample
The light path schematic diagram in the region (including 44 4 layers of substrate 42, buffer layer 45, biological sample 41 and fluid cavity) of product 41.
According to the basic principle of OIRD, under four layer models shown in Fig. 5 a and Fig. 5 b, OIRD detects the base of biochip
Frequency signal delta I (Ω) and frequency-doubled signal Δ I (2 Ω) can be expressed as:
Δ I (Ω)=I0[|r4p||r4s|sin(φ4p-φ0-φ4s)-|r3p||r3s|sin(φ3p-φ0-φ3s)]J1(π)
sin2α (1)
Δ I (2 Ω)=I0[(|r4p|2-|r3p|2)cos2α-(|r4s|2-|r3s|2)sin2α]J2(π) (2)
Wherein I0It is initial beam intensity, | r4p|、|r4s|、|r3p| and | r3s| it is r respectively4p、r4s、r3pAnd r3sAmplitude, φ4p、
φ4s、φ3pAnd φ3sIt is r respectively4p、r4s、r3pAnd r3sPhase (argument)；φ0It is the phase difference that phase-shifter introduces；α is analyzing
The angle of device optical axis, J1(π) and J2(π) is single order and second order Bessel function respectively, and value is respectively 0.2846 and 0.4854.
Referring to Fig. 5 a and Fig. 5 b detailed description above-mentioned formula (1) and (2) in each physical quantity definition, derivation and
The selection of parameter.
As shown in figure 5 a and 5b, λ indicates incident light, r3pAnd r3sIndicate that biochip surface is not bound with biological sample
Reflectivity of 41 region to P and s polarised light, r4pAnd r4sIndicate that biochip surface is combined with the region of biological sample 41 to P
With the reflectivity of s polarised light.Wherein εs、εp、εdAnd ε0Respectively indicate substrate 42, buffer layer 45, biological sample 41 and fluid cavity 44
Dielectric constant, and WithIt is illustrated respectively in substrate 42, buffer layer 45, biological sample 41 and fluid cavity 44
Refraction angle.H is the thickness of buffer layer 45, and d is the thickness of biological sample 41.
In being not bound in the region of biological sample 41 for biochip surface, the reflectivity r of p and s polarised light3pAnd r3s
It respectively indicates are as follows:
Wherein,WithIt is illustrated respectively in the reflection of substrate 42 and 45 interface of buffer layer to p-polarization light and s polarised light
Rate,WithBuffer layer 45 and 44 interface of fluid cavity are illustrated respectively in the reflectivity of p-polarization light and s polarised light.
In the region of the combination biological sample 41 of biochip surface, the reflectivity r of p and s polarised light4p、r4sTable respectively
It is shown as:
Wherein
WithIt is illustrated respectively in the reflection of buffer layer 45 and 41 interface of biological sample layer to p-polarization light and s polarised light
Rate,WithBiological sample layer 41 and 44 interface of fluid cavity are illustrated respectively in the reflectivity of p-polarization light and s polarised light.
It is generally ε by dielectric constantaAnd εbThe interface that constitutes two kinds of materials a and b can be with to the reflectivity of p and s polarised light
It is provided by following formula:
WhereinWithRespectively refraction angle of the incident light in a, b two media.
Therefore according to formula (7) and (8), the dielectric constant for substituting into specific medium can calculate each interface to p and s polarised light
Reflectivity.In other words, it that is, can be obtained with formula (7) and (8):
Formula (3)-(6) are updated to formula (1) and (2) by us, ignore initial beam intensity I0, and corresponding parameter is given,
OIRD fundamental frequency and frequency-doubled signal intensity can be calculated with the changing rule of 45 thickness of buffer layer.
For example, choosing the silicon wafer of polishing as substrate 42, SiO in one embodiment according to the present invention2It buffers
Layer 45, wavelength X=632.8nm of exploring laser light detects the incidence angle of lightThe refractive index n of silicon substrate 2s=3.88+
0.02i, the refractive index n of fluid cavity 4 (air)0=1, SiO2The refractive index n of buffer layer 45p=1.5, the refraction of biological sample 41
Rate is 1.52.The phase difference of phase-shifter takes φ in OIRD detection device0=φ3p-φ3s, angle [alpha]=45 ° of analyzer.Biological sample
Thickness d=1nm of product 41.
Above-mentioned parameter is brought into formula (1) and (2) it is found that working as SiO2The thickness h of buffer layer 45 is changed to from 0
When, the situation of change of fundamental frequency signal intensity and frequency-doubled signal intensity in OIRD signal is as shown in figures 6 a and 6b.According to buffering
The difference of 45 thickness of layer, the fundamental frequency signal intensity and frequency-doubled signal intensity of OIRD change between negative maximum and positive maximum.
As shown in Figure 6 a, if the thickness h of buffer layer 45 is 0 and 256nm, the intensity of fundamental frequency signal reaches negative very big
Value.If the thickness h of buffer layer 45 is 128nm, the intensity of fundamental frequency signal reaches positive maximum.And work as the thickness of buffer layer 45
When respectively 90 and 160nm, the intensity of fundamental frequency signal is 0.
As shown in Figure 6 b, if the thickness h of buffer layer 45 is 160nm, the intensity of frequency-doubled signal reaches positive maximum.?
When the thickness h of buffer layer 45 is 90nm, the intensity of frequency-doubled signal reaches negative maximum.When 45 thickness hs of buffer layer are respectively
When 0,128 and 250nm, the intensity of frequency-doubled signal is 0.
It can be seen that those skilled in the art can select according to actual needs suitable buffer layer 45 material and its
Thickness h, to obtain maximum frequency-doubled signal intensity or maximum fundamental frequency signal intensity, thus improve OIRD detection sensitivity and
Resolution ratio.Or can be by the thickness h of the suitable buffer layer 45 of selection, and it is strong to obtain the preferable frequency-doubled signal of intensity simultaneously
Degree and fundamental frequency signal intensity, wherein being preferably, the intensity of fundamental frequency signal is more than its negative maximum or the 20% of its positive maximum, spy
Not You Xuandichaoguo 50%, and the intensity of frequency-doubled signal is more than the 20% of its negative maximum or its positive maximum, particularly preferably
More than 50%.Such as the thickness h of buffer layer 45 is certain special thickness such as 55nm or 140nm, so that it may while it is strong to obtain signal
Ideal fundamental frequency and frequency multiplication two paths of signals are spent, thus while improving the sensitivity and resolution ratio of OIRD detection, also
The certain physical characteristics of detected biological sample can be fitted according to two paths of signals.
The biochip based on " three layer model " being mentioned above is equivalent in the biochip based on " four layer models "
The case where buffer layer 45 is about zero.As shown in figure 6 a and 6b, when buffer layer 45 be about zero when (be mentioned above based on " three
The biochip of layer model "), the intensity of frequency-doubled signal is zero, and can only obtain fundamental frequency signal.Accordingly, with respect to based on three
The biochip of layer model, two paths of signals can be obtained simultaneously by being somebody's turn to do the biochip based on four layer models.Furthermore it is also possible to pass through tune
The thickness of buffer layer 45 is controlled, and obtains preferable fundamental frequency signal value and/or frequency-doubled signal value.
According to another aspect of the invention, additionally provide it is a kind of for detect it is according to the present invention based on opaque substrate and
The OIRD detection method of the biochip of " four layer models ".
It, all must be to fundamental frequency before detection in OIRD detection method in the prior art based on transparent substrates and " three layer model "
With frequency-doubled signal while back end " zeroing ".However, in the feelings using the biochip according to the present invention based on " four layer models "
Under condition, before OIRD detection, it is not necessary to return to zero simultaneously to fundamental frequency and frequency-doubled signal.
Actual OIRD detection in, value detected by the photodetector in OIRD detection device be OIRD signal with
The superposition of back end signal.In order to keep testing result more accurate, when back end signal and OIRD signal are positive value or are negative value,
It would generally be returned to zero by back end, keep the absolute value of back end signal as small as possible, to improve signal-to-noise ratio.
However, for back end signal and OIRD signal be one just, one it is negative when, if the absolute value of back end signal is too small, meeting
The amplitude of OIRD signal is caused to reduce, it is unfavorable to the sensitivity of detection instead.This is because the photoelectricity in OIRD detection device is visited
The size that device is merely able to detection signal absolute value is surveyed, and signal can not be detected for positive value or negative value.Therefore, when back end signal
After OIRD Signal averaging, due to positive and negative counteracting, the amplitude of OIRD signal can reduce.Such as when back end signal is 0.6mV, OIRD
When signal is -1mV, the absolute value of both signal detected by photodetector is superimposed signal, i.e. 0.6mV.At this time
Value detected by photodetector changes between 0.6mV and 0.4mV.And at this point, if back end signal value is increased to
1mV, value detected by photodetector changes between 1mV and 0mV at this time, it is seen that " not returning to zero " can make the width of OIRD signal
Degree significantly increases.
Under normal conditions, back end signal is positive value, and the existing biochip based on " three layer model " is obtained
OIRD signal is also usually positive value, therefore in OIRD detection method in the prior art, same to fundamental frequency and frequency-doubled signal before detection
When back end " zeroing " be beneficial.
However for the biochip according to the present invention based on opaque substrate and " four layer models ", such as Fig. 6 a and
Shown in Fig. 6 b, fundamental frequency signal and frequency-doubled signal differ 90 degree in phase, that is to say, that buffer layer 45 is in some thickness conditions
Under, fundamental frequency and frequency-doubled signal may be positive or negative simultaneously, and buffer layer 45 is under other thickness conditions, fundamental frequency and frequency-doubled signal
It may be the two paths of signals of positive and negative reverse phase.Either fundamental frequency or frequency multiplication do not need not only " to adjust when its signal is negative value
Zero ", privately signal should be transferred to certain big amplitude instead, just can guarantee that the signal of needs is not cancelled and subtracts in this way
It is small.
In the OIRD detection method provided according to the present invention, using the above-mentioned biochip with buffer layer 45, into
Before the formal OIRD detection of row, first back end signal is debugged, when the intensity and signal-to-noise ratio of OIRD signal obtained are equal
When reaching maximum value, the value of corresponding back end signal is the optimum value of back end signal.The setting of back end signal is best at this
Value or at least close to the optimum value after, start OIRD detection.Detection in the prior art can be used in the specific steps of OIRD detection
Step is after progress.
Referring to described in wherein " close to the optimum value ", back end signal is arranged to, so that oblique incident ray difference in reflection detects
The intensity of the signal of method is at least up to the 60% of maximum of intensity.
In addition, the silicon and 32,42 pairs of detection light of germanium substrate due to biochip provided by the invention are opaque, biology
Chip need to be placed in above fluid cavity, and be sealed using 36,46 pairs of fluid cavitys of transparent window, and through detection light.And by
In increasing window 36,46 on fluid cavity, the reflected beams on two surfaces up and down of window 36,46 are also just added additional.
Therefore, in order to exclude window 36,46 the reflected beams on two surfaces up and down interference, existing OIRD shown in FIG. 1 can be examined
The distance between analyzer 19 and the photodetector 120 surveyed in device increase, and increase an aperture and one between
A lens, thus only make biological sample 31, the reflected light on 41 surfaces enters photodetector 120, improve OIRD detection it is sensitive
Degree and resolution ratio.
According to another aspect of the present invention, the method for making biochip according to the present invention is additionally provided.
(1), the method for biochip of the manufacture based on " three layer model ", comprising:
1) substrate is done with the silicon of surface polishing or germanium, forms active layer on the silicon or germanium substrate surface, which can be with
For epoxy group, an aromatic plant metioned in ancient books base or polymer etc.；
2) biological sample is prepared on active layer, so that biological sample is fixed on substrate.
(2), the method for biochip of the manufacture based on " four layer models ", comprising:
1) substrate is done with the silicon of surface polishing or germanium；
2) buffer layer is formed on the substrate, wherein the thickness of buffer layer according to above-mentioned formula (1) and the calculated result of (2) and
Setting.By selecting the thickness of buffer layer, fundamental frequency and frequency multiplication two paths of signals, or the thickness by selecting buffer layer can be obtained simultaneously
The intensity of degree, the fundamental frequency or frequency-doubled signal that may make OIRD signal obtains maximum value；
3) active layer is formed on the buffer layer；
4) biological sample is prepared on active layer.
Wherein, if the material selection of buffer layer has the material of active layer characteristic, buffer layer can play active layer
Effect (i.e. immobilizing biological samples), active layer can omit in this case.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with several specific embodiments,
The present invention is described in more detail.
Embodiment 1
According to the method (one) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
Include:
The substrate being made of the monocrystalline silicon piece of surface polishing is provided；
Epoxy-activated layer is made in substrate surface；
The BSA protein biology sample spot of 5 × 10 10mg/mL of point system on the active layer left side, 5 × 10 5mg/ of the right point system
The Mouse IgG biological sample point of mL.
The biochip that this method is obtained is placed on the container with transparent window and is formed by fluid cavity, fluid cavity
Interior filling has the reaction solution of specific binding with Mouse IgG.Wherein the side with biological sample of biochip with should
Transparent window is staggered relatively, so that biological sample be made to be exposed to the detection light of OIRD detection device by the transparent window.
The advantages of in order to illustrate biochip according to the present invention, has also made the life using glass slide as substrate
As a comparison case, production method is same as mentioned above for object chip.
It after biochip is cleaned, is detected respectively with biochip of the OIRD device to above two various substrates, respectively
Corresponding testing result is obtained to be compared.
Fig. 7 a and Fig. 7 b are the two-dimensional scanning figures of testing result, and wherein Fig. 7 (a) is the biological core that glass slide does substrate
The testing result of piece, Fig. 7 (b) are the testing results for the biochip that monocrystalline silicon piece does substrate.It can be with from the comparison of 7a and Fig. 7 b
It is apparent from, the fundamental frequency signal that silicon wafer does the biochip of substrate makees the fundamental frequency signal of the biochip of substrate than glass slide
5 times are enhanced, the frequency-doubled signal that silicon wafer makees the biochip of substrate makees the frequency multiplication letter of the biochip of substrate than glass slide
Number enhance 3 times.
The experimental results showed that the biochip according to the present invention using silicon as substrate, is greatly improved OIRD signal
Intensity, effectively improve OIRD detection sensitivity and resolution ratio.
Embodiment 2
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
Include:
The substrate being made of the monocrystalline germanium wafer of surface polishing is provided；
SiO is formed on the substrate2Buffer layer；
Buffer-layer surface production about 1nm thickness an aromatic plant metioned in ancient books base or epoxy-activated layer with immobilizing biological samples；
Biological sample is made on active layer.
The biochip that this method is obtained is placed on the container with transparent window and is formed by fluid cavity, fluid cavity
It is interior to be filled with reaction solution.Wherein the side with biological sample of biochip and the transparent window are staggered relatively, to make
Biological sample is exposed to the detection light of OIRD detection device by the transparent window.
Wherein the thickness of buffer layer is according to the above formula (1) about " four layer models ", the calculated result of (2)
And it designs.For example, for the present embodiment, wavelength X=632.8nm of exploring laser light used by OIRD device detects light
Incidence angleThe refractive index n of germanium substrates=5.47+0.82i, SiO2The refractive index n of buffer layerp=1.5, fluid cavity
The refractive index of interior liquid (reaction solution) is approximately equal to the refractive index n of water0=1.33, the refractive index of biological sample is 1.52.
The phase difference of phase-shifter takes φ in OIRD detection device0=φ3p-φ3s, angle [alpha]=45 ° of analyzer.The thickness of biological sample 1
D=1nm.It brings above-mentioned parameter into formula (1), in (2), SiO can be calculated2Buffer layer thickness h from 0 increase to 300nm when,
OIRD fundamental frequency and frequency-doubled signal with h variation relation.
Fig. 8 a and 8b respectively illustrate the biochip that substrate is done for germanium, the fundamental frequency and frequency-doubled signal in OIRD signal with
SiO2The situation of change of buffer layer thickness.It can be seen that from Fig. 8 a when thickness h is 0 or 250nm, fundamental frequency signal reaches cathode
Big value；When thickness h is 125nm, fundamental frequency signal reaches positive maximum；When thickness h is 95nm, frequency-doubled signal reaches negative maximum
Value；When thickness h is 155nm, frequency-doubled signal reaches positive maximum；When thickness h is 85 or 170nm, fundamental frequency signal 0,
When thickness h is 0,125 or 250nm, frequency-doubled signal 0.
Therefore, the thickness of buffer layer can be chosen according to the calculated result.
In the present embodiment 2, SiO2Buffer layer is selected as 125nm, so that finally obtained biochip can obtain
Optimal OIRD fundamental frequency signal is obtained, i.e. OIRD fundamental frequency signal reaches positive maximum.Embodiment 3
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, SiO2The thickness of buffer layer is selected as
95nm。
As shown in Figure 8 b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e., and OIRD times
Frequency signal reaches negative maximum.
Embodiment 4
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, SiO2The thickness of buffer layer is selected as
250nm。
As shown in Figure 8 a, the biochip that the present embodiment obtains can obtain optimal OIRD fundamental frequency signal, i.e. OIRD base
Frequency signal reaches negative maximum.
Embodiment 5
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, SiO2The thickness of buffer layer is selected as
155nm。
As shown in Figure 8 b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e. positive maximum
The OIRD frequency-doubled signal of value, preparation can obtain the biochip of OIRD frequency-doubled signal positive maximum.
Embodiment 6
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, SiO2The thickness of buffer layer is selected as
55nm。
As shown in figs. 8 a and 8b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.004, about the 55% of fundamental frequency signal intensity negative maximum, the intensity of frequency-doubled signal
About 0.003, about about the 60% of frequency-doubled signal intensity negative maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Embodiment 7
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, SiO2The thickness of buffer layer is selected as
140nm。
As shown in figs. 8 a and 8b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.007, about the 70% of fundamental frequency signal intensity positive maximum, the intensity of frequency-doubled signal
About 0.004, about about the 80% of frequency-doubled signal intensity positive maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Embodiment 8
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 2 are essentially identical, the difference is that, substrate by surface polishing monocrystalline silicon piece structure
At being not filled by reaction solution in fluid cavity, and be only filled with air, in addition SiO2The thickness of buffer layer is selected as 128nm.
The structural parameters and test parameter of biochip provided in this embodiment meet reality corresponding to Fig. 6 a and Fig. 6 b
The structural parameters and test parameter of example are applied, therefore the thickness of the buffer layer in the present embodiment can refer to described in Fig. 6 a and Fig. 6 b
Changing rule and be designed.
In the present embodiment, SiO2The thickness of buffer layer is designed to 128nm.As shown in Figure 6 a, the present embodiment obtains
Biochip can obtain optimal OIRD fundamental frequency signal, i.e. OIRD fundamental frequency signal reaches positive maximum.
Embodiment 9
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 8 are essentially identical, the difference is that SiO2The thickness of buffer layer is selected as
90nm。
As shown in Figure 6 b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e., and OIRD times
Frequency signal reaches negative maximum.
Embodiment 10
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 8 are essentially identical, the difference is that SiO2The thickness of buffer layer is selected as
160nm。
As shown in Figure 6 b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e., and OIRD times
Frequency signal reaches positive maximum.
Embodiment 11
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 8 are essentially identical, the difference is that SiO2The thickness of buffer layer is selected as
55nm。
As shown in figure 6 a and 6b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.003, about the 80% of fundamental frequency signal intensity negative maximum, the intensity of frequency-doubled signal
About 0.0025, about about the 55% of frequency-doubled signal intensity negative maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Embodiment 12
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 8 are essentially identical, the difference is that SiO2The thickness of buffer layer is selected as
140nm。
As shown in figure 6 a and 6b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.003, about the 50% of fundamental frequency signal intensity positive maximum, the intensity of frequency-doubled signal
About 0.003, about about the 65% of frequency-doubled signal intensity positive maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Embodiment 13
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
Include:
The substrate being made of the monocrystalline silicon piece of surface polishing is provided；
Buffer layer is formed on the substrate, which is made of the polymer with binding biomolecules characteristic, such as poly-
There is binding biomolecules characteristic in other words to constitute material of buffer layer itself for ethylene, the material due to constituting buffer layer
It is a kind of activation layer material, therefore fixed function effectively can be generated to biological sample, therefore active layer need not be additionally set again；
Biological sample is made on the buffer layer.
The biochip that this method is obtained is placed on the container with transparent window and is formed by fluid cavity, fluid cavity
It is interior to be filled with reaction solution.Wherein the side with biological sample of biochip and the transparent window are staggered relatively, to make
Biological sample is exposed to the detection light of OIRD detection device by the transparent window.
Wherein the thickness of buffer layer is according to the above formula (1) about " four layer models ", the calculated result of (2)
And it designs.For example, for the present embodiment, wavelength X=632.8nm of exploring laser light used by OIRD device detects light
Incidence angleThe refractive index n of silicon substrate 2s=3.88+0.02i, the refractive index n of polymer buffer layerp=1.54, stream
The refractive index of liquid (reaction solution) in body cavity 4 is approximately equal to the refractive index n of water0=1.33, the refractive index of biological sample 1 is
1.52.The phase difference of phase-shifter takes φ in OIRD detection device0=φ3p-φ3s, angle [alpha]=45 ° of analyzer.Biological sample
Thickness d=1nm.Bring above-mentioned parameter into formula (1), in (2), can be calculated buffer layer thickness h from 0 increase to 300nm when,
The relationship that OIRD fundamental frequency and frequency-doubled signal change with h.
Fig. 9 a and 9b are shown respectively for above-mentioned biochip, OIRD fundamental frequency and frequency-doubled signal with polymer buffer thickness degree
The case where h changes.It can be seen that from Fig. 9 a when thickness h is 0 or 250nm, fundamental frequency signal reaches negative maximum；It is in thickness h
When 125nm, fundamental frequency signal reaches positive maximum；It can be seen that from Fig. 9 b when thickness h is 95nm, frequency-doubled signal reaches cathode
Big value；When thickness h is 155nm, frequency-doubled signal reaches positive maximum；When thickness h is 85 or 170nm, fundamental frequency signal 0,
When h is with a thickness of 0,125 or 250nm, frequency-doubled signal 0.
Therefore, the thickness of buffer layer can be chosen according to the calculated result.
In the present embodiment 13, polymer buffer layer is selected as 125nm, so that finally obtained biochip can
Optimal OIRD fundamental frequency signal is obtained, i.e. OIRD fundamental frequency signal reaches positive maximum.
Embodiment 14
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 13 are essentially identical, the difference is that the thickness of polymer buffer layer is selected as
95nm。
As shown in figure 9b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e., and OIRD times
Frequency signal reaches negative maximum.
Embodiment 15
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 13 are essentially identical, the difference is that the thickness of polymer buffer layer is selected as
250nm。
As illustrated in fig. 9, the biochip that the present embodiment obtains can obtain optimal OIRD fundamental frequency signal, i.e. OIRD base
Frequency signal reaches negative maximum.
Embodiment 16
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 13 are essentially identical, the difference is that the thickness of polymer buffer layer is selected as
155nm。
As shown in figure 9b, the biochip that the present embodiment obtains can obtain optimal OIRD frequency-doubled signal, i.e., and OIRD times
Frequency signal reaches positive maximum.
Embodiment 17
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 13 are essentially identical, the difference is that the thickness of polymer buffer layer is selected as
55nm。
As illustrated in figures 9 a and 9b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.003, about the 60% of fundamental frequency signal intensity negative maximum, the intensity of frequency-doubled signal
About 0.003, about about the 65% of frequency-doubled signal intensity negative maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Embodiment 18
According to the method (two) of above-mentioned production biochip, a kind of manufacturing method of biochip is present embodiments provided,
This method and method described in embodiment 13 are essentially identical, the difference is that the thickness of polymer buffer layer is selected as
140nm。
As illustrated in figures 9 a and 9b, the biochip that the present embodiment obtains can obtain OIRD fundamental frequency and frequency multiplication two-way letter simultaneously
Number, wherein the intensity of fundamental frequency signal is about 0.001, about the 20% of fundamental frequency signal intensity negative maximum, the intensity of frequency-doubled signal
About 0.0035, about about the 75% of frequency-doubled signal intensity negative maximum.
It can be seen that it is relatively strong to can get intensity by selecting the thickness of buffer layer for biochip provided in this embodiment
Fundamental frequency and frequency multiplication two paths of signals.
Other embodiments according to the present invention, wherein the material for constituting buffer layer is not limited to the SiO in above-described embodiment2
And polymer, such as can also be SrTiO3, LaAlO3It equal transparent materials and other are suitable as activation layer material or are suitable for
In conjunction with the transparent polymer etc. of biological sample.In addition, for the buffer layer material that can generate enough adhesive force to biological sample
Expect, the active layer on buffer layer also can be omitted.
In addition, term " transparent " described in the present invention, refers to transparent or semitransparent to the wavelength of detection light.
The silicon substrate and germanium substrate used in the present invention, can also be with a variety of crystal habits, such as monocrystalline or polycrystalline.Silicon substrate
50 angstroms are preferably less than with the surface average roughness of germanium substrate.
To sum up, the present invention provides a kind of biochip based on silicon substrate or germanium substrate, is greatly improved OIRD signal
Intensity.
Further, according to an aspect of the invention, there is provided one kind is based on the biochip of " four layer models ", in silicon
Or a buffer layer is set between substrate and biological sample.The first interface, buffer layer and biological sample are formed between buffer layer and substrate
Second contact surface is formed between product.Through theoretical calculation it is found that due to caused by the reflected light on first interface and second contact surface
Interference effect makes intensity all generating periods with the change of buffer layer thickness of the fundamental frequency and frequency-doubled signal in OIRD signal
Variation.In other words, with the difference of buffer layer thickness, the intensity of OIRD fundamental frequency and frequency-doubled signal can in positive maximum and
Change between negative maximum.Therefore, by the way that the thickness of buffer layer is reasonably arranged, the fundamental frequency or frequency multiplication letter of OIRD signal can be made
Number intensity obtain maximum value, to improve the detection sensitivity and resolution ratio of OIRD detection method.Or by reasonably setting
The thickness of buffer layer is set, can obtaining fundamental frequency and frequency multiplication two paths of signals simultaneously, (preferably the intensity of fundamental frequency signal is more than its negative maximum
The 20% of value or its positive maximum, while the intensity of frequency-doubled signal is more than its negative maximum or the 20% of its positive maximum), and root
The certain physical characteristics of detected biological sample can be fitted according to two paths of signals.This is the biology currently based on " three layer model "
Chip (can only obtain fundamental frequency signal all the way) cannot be achieved.
In addition, present invention is alternatively directed to the biochips based on " four layer models " to provide a kind of OIRD without back end zeroing
Detection method can further improve the intensity of OIRD signal.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Although ginseng
It is described the invention in detail according to embodiment, those skilled in the art should understand that, to technical side of the invention
Case is modified or replaced equivalently, and without departure from the spirit and scope of technical solution of the present invention, should all be covered in the present invention
Scope of the claims in.
Claims (10)
1. a kind of biochip for oblique incident ray reflection difference detection method, comprising:
Substrate is made of silicon or germanium；
Fixed to the biological sample on the substrate；
Wherein also there is between the substrate and the biological sample transparent buffer layer, select the thickness of the buffer layer, so that
It obtains in oblique incident ray reflection difference detection method, can:
I) fundamental frequency signal and frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method are obtained simultaneously；Or
Ii) make the intensity of the fundamental frequency signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or its
Positive maximum；Or
Iii) make the intensity of the frequency-doubled signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or
Its positive maximum.
2. biochip according to claim 1 further includes the container to form fluid cavity, which has transparent window,
The biological sample is located in the fluid cavity, and the biological sample is exposed to oblique incident ray by the transparent window and reflects
Light is detected used in difference detection method.
3. biochip according to claim 1, the biological sample is fixed on the substrate by active layer.
4. biochip according to claim 1, wherein the thickness of the buffer layer is selected as making the fundamental frequency signal
Intensity be more than the 20% of its negative maximum or its positive maximum, while the intensity of the frequency-doubled signal being made to be more than its negative maximum
Or the 20% of its positive maximum.
5. biochip according to claim 1, wherein the material of the transparent buffer layer is SiO2、SrTiO3、LaAlO3
Or polymer.
6. biochip according to claim 1, wherein the biological sample is fixed to the buffer layer by active layer
On.
7. biochip according to claim 1, wherein the buffer layer is by the activation suitable for the fixed biological sample
Material is constituted, and the biological sample is on the buffer layer.
8. a kind of oblique incident ray reflects difference detection method, for detecting biological core described in any one of -7 according to claim 1
Piece, which comprises
I) fundamental frequency signal and frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method are obtained simultaneously；Or
Ii) make the intensity of the fundamental frequency signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or its
Positive maximum；Or
Iii) make the intensity of the frequency-doubled signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or
Its positive maximum.
9. a kind of for detecting the oblique incident ray difference in reflection detection side of biochip described in any one of -7 according to claim 1
Method, comprising:
A predetermined value is set by the value of back end signal, which makes the fundamental frequency signal of oblique incident ray reflection difference detection method
Or the intensity of frequency-doubled signal is at least up to the 60% of the maximum intensity that the intensity of the fundamental frequency signal or frequency-doubled signal can reach；
The detection of oblique incidence light reflection difference method is carried out to the biochip.
10. a kind of method for manufacturing biochip described in any one of -7 according to claim 1, comprising:
The substrate being made of silicon or germanium is provided；
Transparent buffer layer is formed on the substrate；
The immobilizing biological samples on the transparent buffer layer,
The thickness of the buffer layer is selected, so that in oblique incident ray reflection difference detection method, can:
I) fundamental frequency signal and the frequency-doubled signal in the signal of the oblique incident ray reflection difference detection method are obtained simultaneously；
Or
Ii) make the intensity of the fundamental frequency signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or its
Positive maximum；Or
Iii) make the intensity of the frequency-doubled signal in the signal of oblique incident ray reflection difference detection method be equal to its negative maximum or
Its positive maximum.
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