CN104568907B - Micro-fluidic SERS chip for nondestructive testing of blood and biological sample - Google Patents
Micro-fluidic SERS chip for nondestructive testing of blood and biological sample Download PDFInfo
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- CN104568907B CN104568907B CN201510030105.6A CN201510030105A CN104568907B CN 104568907 B CN104568907 B CN 104568907B CN 201510030105 A CN201510030105 A CN 201510030105A CN 104568907 B CN104568907 B CN 104568907B
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Abstract
The invention provides a micro-fluidic SERS chip for nondestructive testing of blood and a biological sample. The chip is in a 'sandwich' structure which is formed by a substrate, a PDMS interlayer and a cover plate, and comprises a micro-channel, a sampling opening and a discharge opening, wherein a plurality of detection holes are formed in the middle of the micro-channel to form a porous detection zone. A dual-layer nano SERS enhancement base which can be repeatedly used is prepared in an in-situ manner by combination of silver mirror reaction and a chemical plating self-assembly method, so that the SERS detection sensitivity and the detection efficiency are effectively improved. The sampling opening is designed into a conical end sampling opening to form a capillary sampling mode; a to-be-detected liquid is directly drained to a to-be-detected zone by the capillary action; the effects caused by the dripping process are avoided; the chip comprises a plurality of detection zones, so that once sampling and multi-time detection can be achieved; and high detection repeatability is improved by determining the average value. The chip is relatively small in size, and convenient to carry, and can be used as a detection tool which is carried by a tester, and is relatively simple in preparation method, low in cost and suitable for multi-point parallel testing of the biological sample.
Description
Technical field
The present invention relates to biochemistry detection, chip, spectral technique field.
Background technology
Dynamic Non-Destruction Measurement is not destroyed and damages the performance of examined object, has weight in special biochemistry detection
Want meaning.At present, the detection meanss for being usually used in blood and biological specimen include GC/LC-MS, DNA tests, immunoassay and micro-
Observation etc., these detection modes consumption sample sizes are larger and have damage to sample.The method of existing Non-Destructive Testing is mainly adopted
The sound and light detecting patterns such as ultrasound detection, ray detection, Magnetic testi, acoustic emission detection, laser hologram detection, infrared detection, but this
A little technologies are mainly used in the larger solid detection of mechanical devices or bone isodensity, and Raman spectroscopy is as which is in inspection
The damage of chemistry and machinery will not be caused during survey, analysis to sample, is not easy to produce light and heat decomposition situation, simultaneously because
The Raman scattering of water is very weak so as to has special advantage in the detection of bio-chemical solution class sample, is very suitable for health care
Field is particularly the special sample Non-Destructive Testings such as blood.
Due to normal Raman spectroscopy signal it is weaker, it is actually detected in would generally strengthen the drawing of test substance with SERS substrates
Graceful response signal, so as to reduce detection limit, improves detection sensitivity.SERS strengthens substrate and is broadly divided into suspended state colloidal sol and solid
Ordered media, the former be by SERS suspended states colloidal sol mix with liquid to be detected then together with drop in the tables such as quartz, silicon chip, glass
Face is tested, and its measuring stability and repeatability are poor, destruction is easily produced to sample and is polluted；And the latter's SERS solids are orderly
Medium is then that testing sample is added drop-wise to its surface to be tested, its on prepared by nanoreinforcement material with compared with more options, together
When test stability and repeatability on have a clear superiority.So, develop a kind of micro-fluidic SERS of closing Non-Destructive Testing
Chip detection new equipment is necessary." highly sensitive, high repeatability surface enhanced draws 103487426 A of Patents documents CN
The detection method and device of graceful spectrum " discloses one kind can be by driving means Rotary tray, by testing liquid even spread
On the surface of SERS substrates, the device of multiple spot Raman test is carried out, but, the nano material in the device is adopted and is applied directly to
Prepared by ground glass, sand paper, filter paper or lens paper, nanostructured is combined not tight with detector substrate, it is impossible to reused；Separately
Outward, adopt motor as driving in device, make whole device huger, be inconvenient to focus in detection, increased cost, disappear
Consume and use difficulty." a kind of SERS probe molecules are from collection micro-pipe and preparation method thereof for 103774088 A of patent documentation CN
And application " disclose a kind of by inside pipe wall surface Pt catalysis H2O2The oxygen of decomposition overflows generation pressure differential and obtains a driving
Power, so as to realize SERS probe molecules from the micro-tubular structure collected, but, the device is present destruction and stable testing to sample
Property difference the problems such as.
At present, also have in microchannel and make fixed SERS substrates, mainly using microelectromechanical systems（Micro
Electromechanical System, MEMS）Process technology is prepared or is prepared using physico-chemical methods such as Chemical self-assemblies.The former
Have the advantages that nanometer particle size is controllable in order, but strengthen host material species limitation greatly and prepare high processing costs；The latter has
Have matrix species easily to expand, processing cost is relatively low, and microfluidic channel associated methods it is flexible the features such as.In Chemical self-assembly legal system
During standby SERS substrates, the existing research for preparing nanometer gold and silver and method are more, but are all such underlying structures in SERS
Need to improve in the loss of signal and detection sensitivity.
The content of the invention
It is an object of the invention to provide a kind of blood and biological specimen Non-Destructive Testing are with micro-fluidic SERS chips, by
Silver mirror reaction film forming is adopted in micro-fluidic chip detection zone, and then combines chemical plating self-assembling method, it is former on compound chip
Good stability, reusable double-layer nanometer metal SERS substrates are prepared in position, effectively improve the sensitivity of blood testing
And repeatability, and further testing liquid is directly drained into region to be measured using capillarity, is kept away by the design of microfluidic circuit
Exempt from the impact that Deca process is caused, on chip, there are multiple detection zones, realize single injected sampling, repeated detection, to improve detection
High duplication, realizes the high throughput testing of sample.
Technical scheme is as follows：
A kind of blood and biological specimen Non-Destructive Testing with micro-fluidic SERS chips, for serum/lossless inspection of blood/body fluid
Survey." sandwich " structure that chip is constituted using substrate-PDMS interlayers-cover plate, include in which microchannel and one end injection port,
The outlet of the other end, is provided with multiple detection holes in the middle of microchannel, forms porous detection zone.It is formed with the microchannel and receives
Rice SERS strengthens substrate, and the forming method that nanometer SERS strengthens substrate is as follows：First with silver mirror reaction in microchannel surface
Plating Ag films；Then passing to polycation electrolyte PDDA makes Ag films surface positively charged；By electrostatic force Chemical self-assembly
Pattern, polytype metal nanoparticles such as negatively charged different size, the Au of different-shape or Ag or Cu are deposited on
Ag films surface；Finally, with chemical plating method on nanoparticle in-situ deposition, i.e., in microchannel formed nanometer SERS strengthen base
Bottom.
Further, the chip profile of injection port position is designed as taper by the present invention, is formed tapered end injection port, is hair
Tubule sample introduction pattern.
Based on micro-fluidic SERS analysis chips microdevice proposed by the present invention, blood and biological specimen inspection are carried out with this device
The method of survey is：Directly contacted with blood and biological specimen etc. with the tapered end injection port of chip microdevice, testing sample passes through hair
Spy reaches detection zone circular hole with along micro passage；Chip is placed on into Raman detector object stage, when adjusting optical maser wavelength, exposure
Between, cycle-index, laser power, the depth of focus, test obtain Raman spectrum；It is right in the range of sample to have in SERS substrates
Sample carry out multiple spot detection, using obtain multiple spot testing sample spectral information.
SERS detections are carried out using the micro-fluidic SERS chip-detecting apparatus of the present invention, is conducive to improving blood and biology
The test limit of pattern detection, sensitivity, repeatability and operability.
The pattern of plated film and self assembly is present invention employs, is dexterously assembled using silver mirror reaction produced film silver again and is received
The Double layer high efficient SERS substrates of meter Jin etc., are facilitated using silver mirror reaction film forming and fast, silver medium SERS effects are good and silverskin hinders
Only light projection improves the advantage of sample detection signal, then in silverskin over-assemble nano-metal particle, can effectively improve SERS inspections
Survey sensitivity and detection efficiency.
Injection port is designed to tapered end injection port by the present invention, forms capillary sample inlet pattern, will be to be measured using capillarity
Liquid directly drains into region to be measured, using capillarity sample introduction, the loss produced in being prevented effectively from traditional sample introduction and pollution.
The detection zone of the chip includes multiple detection cells, can realize single injected sampling, and repeated detection averages to carry
The high duplication of high detection, can meet high-throughout testing requirement, and greatly shorten detection time, lift detection efficiency.
Not only size is less for the chip, is easy to carry, the checking tool that can be carried with as testing staff, and makes
Preparation Method is relatively easy, with low cost, is suitable to the multipoint parallel test of biochemical sample.The composite can be widely applied to medical hygiene
Field is for patient and blood and the detection of biological specimen of normal person, there is provided the SERS spectrograms of acquisition, can analyze and parse and be each
Each biomacromolecule, the classification of small molecule and content in blood and biological specimen is planted, so as to reference being provided for clinical diagnosises and being referred to
Lead.
Description of the drawings
Figure 1A is the substrate of chip, the PDMS pieces with microchannel and porous detection zone, cover glass exploded view.
Figure 1B is the plane graph that integrated nanometer SERS strengthens substrate microfluidic analysis test chip；
Fig. 2 is to strengthen the people that the test of substrate microfluidic analysis test chip is obtained using integrated nanometer SERS in embodiment two
Whole blood surface enhanced raman spectroscopy figure.
Specific embodiment
Embodiment 1
It is as follows that the micro-fluidic SERS chips of Ag/Au double-metal layer SERS active-substrates prepare specific implementation step：
（1）Prepare a substrate, the substrate and PDMS microchannels, PDMS film are brought into close contact into composition " sandwich " sandwich
Chip structure；
（2）Using silver mirror reaction in microchannel Ag films on plated surface；
（3）Using Chemical self-assembly in Ag film surface-assembled Au nanoparticles；
（4）With chemical plating method on Au nanoparticles in-situ deposition, integrated preparation Ag/Au double-metal layers SERS active groups
Bottom.
In the present embodiment, step（2）Include pretreatment and the silver mirror reaction of glass substrate.
Take glass substrate to be immersed in hot NaOH solution, be cleaned by ultrasonic about 10 minutes with ultrasonic cleaner, to remove
The greasy dirt on its surface.Then it is immersed in 80 DEG C fresh " Piranha washing liquid "（98%H2SO4: 30%H2O2,3:1,v/v）Middle 30min is cold
But room temperature is arrived, then deionized water is rinsed, standby.
Taking the silver nitrate solution that 10ml mass fractions are 2% and mass fraction being added dropwise in small beaker, while stirring be
2% ammonia, when precipitation is wholly absent just, stops Deca weak ammonia, now using alkaline pH reagent paper by solution pH value
It is adjusted to 9（Alkalescence）, then the glucose solution that 2mL mass fractions are 5% is added in small beaker, it is abundant with Glass rod
Stirring makes its mix homogeneously.Then above-mentioned solution is injected in microchannel prepared by substrate and PDMS film combination, this is micro- logical
Road carries out heating in water bath in being put into warm water, water temperature is controlled at 60 DEG C, takes out after reaction 10min, then successively with weak ammonia, go
Ionized water is cleaned, standby.
In the present embodiment, step（3）Include that PDDA changes Ag surface electrical behavior and the chemistry assembling of Au nanoparticles.
PDDA changes Ag surface electrical behavior：0.1% cationic polyelectrolyte PDDA is passed through in the microchannel after silver mirror reaction
30min is stood, then the polyelectrolyte not adsorbed is removed in deionized water flushing microchannel.
The chemistry assembling of Au nanoparticles：It is passed through in microchannel after after 6h, deionized water is rinsed in Au colloidal sols.Au colloidal sols
It is that, according to Lee and Melsel methods, 100mL mass concentrations are 0.01% H4AuCl4Solution stirring is heated to seething with excitement, then plus
The mass concentration for entering 8mL is 1% citric acid three sodium solution, continues agitating heating 15 minutes.Obtained nano particle diameter is in 5-
10nm。
In the present embodiment, step（4）Middle chemical plating：Gold plating liquid is passed through in microchannel（0.2mM hydroxylamine hydrochloride solutions+
0.1% chlorauric acid solution）4min, you can obtain Ag/Au double-metal layer SERS micro-nano structures.
This chip can also adopt other types of metal nano double-decker, and other different types of metal nanos are double-deck
Structure can add proportioning, mixing speed, halide ion addition, illumination condition etc. to realize by changing raw material in experiment.
The micro-fluidic SERS chip structures for preparing are as shown in Figure 1A and Figure 1B：
" sandwich " structure that chip is constituted using substrate 1, PDMS interlayers 2, cover plate 3, includes microchannel 4 and one end in which
Injection port O, the outlet of the other end, be provided with multiple detection holes 5 in the middle of microchannel, form porous detection zone, it is convenient to fix
The position detected every time and the position of detecting instrument launching spot.Double-layer nanometer metal SERS enhancings are integrated with microchannel
Substrate.Chip substrate material is glass, quartz, silicon chip or polymeric material, and patch material is glass, quartz, PDMS（Poly- diformazan
Radical siloxane）Or other transparent polymer films.The PDMS thin layers containing microchannel are bonded on substrate, microchannel size is width
100 ~ 1500um, depth are 30 ~ 100um, 1.4 ~ 1.6cm of length.Above again cladding thickness be the quartz of 1 ~ 3mm, PDMS etc. no
With the cover plate of transparent material, form " sandwich " and decline structure；After preparing nanometer SERS amplified medium films in the original location, only need again
Can be used for SERS detections in Surface mulch transparent cover.
Chip substrate and cover plate thickness are 1mm, and chip includes the microchannel 4 of three parallel arrangeds, and they share one and enter
Sample passage OA, O be injection port position, the long 2 ~ 3mm of OA sections, long 3 ~ 4 mm of OB, OC section, AD 1.4 ~ 1.6cm of length, detection zone circular hole
A diameter of 200 ~ 3000um.
The cuboid of the generally long 1.5cm of chip × wide 1.0cm × high 0.3cm, its chip in injection port position
Profile is designed as taper, forms tapered end injection port 6, and the inclined-plane of tapered end facilitates when sampling and droplet of blood or other bodies into 45 degree of angles
Liquid is close to, and forms capillary sample inlet pattern.Sample to be tested can be introduced from tapered end injection port, be made by the capillary of microchannel
With fluid sample is drained into up to SERS detection zones, without the extra driving of needs.
Embodiment 2
SERS applying detection is carried out to the micro- detectors of SERS in embodiment 1.People's whole blood is passed through using capillary sample introduction micro-
In current sensor, using inVia Raman spectrometers (Reinshaw company of Britain, France), from laser wavelength 785nm, laser
Power 1%, exposure frequency 1 time, time of integration 5s, detection limit 1uL carry out SERS active testings, obtain the SERS spectrums of people's whole blood
Figure（Fig. 2）Two spectral lines are the spectrogram for having SERS substrates and not having SERS substrates to obtain respectively, it can be seen that should from collection of illustrative plates
Micro- detector can be effectively recognized to blood, SERS substrates have obvious reinforced effects to signal.
It can be seen that, the underlying structure of the present invention is reducing SERS signal loss and is improving the advantage that have in detection sensitivity.
Claims (2)
1. with micro-fluidic SERS chips, chip adopts substrate-PDMS interlayers-cover plate structure for a kind of blood and biological specimen Non-Destructive Testing
Into " sandwich " structure, include the outlet of the injection port and the other end of microchannel and one end in which, the injection port is located
The chip profile of position is taper, forms tapered end injection port, is capillary sample inlet pattern；Multiple detections are provided with the middle of microchannel
Hole, forms porous detection zone, has the microchannel of three parallel arrangeds in the chip, and they share a sample intake passage OA, section
Long 2 ~ 3mm, O are injection port position, are capable of achieving the multipoint parallel detection of sample；Nanometer SERS is formed with the microchannel to be strengthened
Substrate, the forming method that nanometer SERS strengthens substrate are as follows：Ag film is plated in microchannel surface first with silver mirror reaction；Then
Being passed through polycation electrolyte PDDA makes Ag films surface positively charged；By electrostatic force Chemical self-assembly pattern, will be with negative
The different size of electric charge, the Au of different-shape or Ag or Cu metal nanoparticles are deposited on Ag films surface；Finally, use chemical plating side
Method in-situ deposition on nanoparticle, i.e., in microchannel forming nanometer SERS strengthens substrate；The silver mirror reaction is by silver-colored ammonia
Solution and mass fraction are 5% glucose solution mix homogeneously, reinject microchannel prepared by substrate and PDMS film combination
In, and complete at a temperature of 60 DEG C of heating in water bath.
2. blood according to claim 1 and biological specimen Non-Destructive Testing are with micro-fluidic SERS chips, it is characterised in that institute
Wide 100 ~ the 1500um in microchannel, deep 30 ~ 100um are stated, a diameter of 200 ~ 3000um in hole is detected.
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| CN104792767B (en) * | 2015-05-08 | 2017-11-17 | 厦门大学 | A kind of micro-fluidic chip and its application method for sol method SERS detections |
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| CN110346350B (en) * | 2019-08-08 | 2020-12-25 | 中国科学技术大学 | SERS structure in microfluidic channel and microfluidic SERS detection method |
| CN112570053A (en) * | 2020-12-07 | 2021-03-30 | 北京航空航天大学 | SERS-SEF dual-mode micro-fluidic chip for glucose detection |
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