CN116660546A

CN116660546A - Tear detecting system

Info

Publication number: CN116660546A
Application number: CN202310634921.2A
Authority: CN
Inventors: 冯云; 李金明; 于昊哲; 杨洲
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-08-29

Abstract

The application relates to a tear detection system, which detects and analyzes tear by using a surface enhanced Raman spectrum technology, the system at least comprises a basal part for collecting tear and a detection part for analyzing tear on the basal part, the basal part drains disease markers in the tear from a collection area to a specific strip according to a specific recognition mode, the detection part carries out qualitative and/or quantitative detection on diseases based on Raman scattering signal changes on the specific recognized strip, wherein a first light beam of the detection part excites Raman scattering signals of the strip on the basal part and transmits the Raman scattering signals to an analysis module of the detection part in a form of a second light beam; according to the application, surface Enhanced Raman Spectroscopy (SERS) and detection test paper are combined, quantitative trace detection can be carried out on dry eye markers and allergy markers at least in a short time under the condition of trace tear sample content, and a plurality of strip detection areas can change the strip modifiers according to the needs, and simultaneously, a plurality of indexes in tear are monitored.

Description

Tear detecting system

Technical Field

The application relates to the technical field of biological fluid detection, in particular to a tear detection system.

Background

Traditional disease diagnosis often requires intravenous blood sampling, and blood collection is a traumatic operation, which can lead to tension on some patients and inaccurate detection. In recent years, efforts have been made to obtain body fluids, such as urine, sweat, saliva, tears, etc., in a noninvasive manner instead of blood for disease diagnosis, with the body fluids being simple in the manner of obtaining a small amount of sample as compared with the blood environment. Wherein tear fluid is a transparent fluid secreted by the lacrimal gland and contains a large amount of water, proteins, lipids, cytokines and metabolites. Proteins in tears, including lactoferrin, lysozyme, secretory immunoglobulin A, lipoproteins, lacrimatory proteins, and lipophiles, are secreted by different cells in the lacrimal gland. Certain protein components of tears are subject to changes in content due to physical disorders (e.g., diabetes, thyroid disorders, and cancer), and thus proteins in tears can be used as biomarkers for diagnosis of specific disorders. For example, tear secreted periostin can be used as a marker for diagnosis of ocular allergic diseases, cytokine Th1 and cytokine Th17 can be used as ocular inflammatory markers, and matrix metalloproteinase MMP-9 can be used as dry eye markers. In addition, glucose in human tears is positively correlated with blood glucose concentration, and diabetes can be monitored by tear glucose detection; small molecule metabolites such as corticosterone, cortisol, etc. in tears can also be used as diagnostic markers for dry eye, so that certain ocular and physical disorders can be detected and monitored by changes in the composition of tears. In addition, tears are not easily polluted by the environment and can be obtained in real time, so that there is a great potential advantage in diagnosing diseases through tears.

Symptoms of allergic conjunctivitis and dry eye behave similarly. Allergic conjunctivitis is a conjunctivitis inflammatory disease caused by type I hypersensitivity and accompanied by subjective symptoms, changes in physical signs, mainly including seasonal allergic conjunctivitis, perennial allergic conjunctivitis, giant papillary conjunctivitis, etc., and diagnosis of ocular allergy by specific immunoglobulin IgE detection or specific inflammatory markers such as interleukin 4 (IL-4), interleukin 5 (IL-5), etc.; dry eye is a kind of ocular disease with dyslacrimation caused by various functional and ocular surface protection mechanism disorders, and is one of the most common ophthalmic diseases in ophthalmic outpatient service, and dry eye patients do not have obvious symptoms at the early stage of dry eye, so many patients suffering from dry eye at the early stage are aggravated by dry eye because they are not detected and treated in time.

One of the more common commercial methods for using tears as an index for diagnosing diseases today is Schirmer test paper, which judges whether dry eye is developed by detecting the amount of tear. If the component content in the tear fluid is to be determined, it is required to detect the component content by liquid chromatography-mass spectrometry.

Since tears of a human body have a relatively low biochemical concentration and an extremely small sample amount, development of an analytical technique with high sensitivity is required to measure tear components of the human body. First, an immunoassay is an analytical method for determining the composition of a sample using an antibody or an antibody-related reagent. Seiffer et al developed a novel immunoassay method that directly quantitatively measured prolactin in human tears; kelly et al report the first microfluidic homogeneous immunoassay method capable of rapidly and specifically qualitatively and quantitatively determining endogenous tear protein biomarkers in human tears. In North America, clinicians typically use an immunodetection-based MMP-9 rapid diagnostic kit to pre-diagnose dry eye patients, in principle using anti-inflammatory drugs early if the patient's tears appear MMP-9 positive. However, the kit has not entered China and can only be used for qualitative detection, and is difficult to follow-up for dry eye patients. For the immunoassay method, the cost of the kit is too high, and the diagnosis of the disease is single. Next, by chromatography-mass spectrometry, grus et al analyzed and compared proteins in tears of dry eye patients with diabetes and non-diabetes and healthy subjects using high performance liquid chromatography, and performed multivariate statistical analysis on the data, found that there were significant differences between the three samples. Huang et al used electrospray assisted laser desorption ionization mass spectrometry to rapidly and sensitively detect the major protein in dry tears. In the current clinical and scientific researches, the liquid chromatography-mass spectrometry is also a gold standard for tear analysis, but due to the large equipment and complex test, a long time is required from tear collection to result analysis, which is extremely inconvenient.

In recent years, with the development of semiconductor laser technology, micro optical fiber spectrometer technology, optical machining technology, and computer science technology, hand-held/portable raman spectrometers are rapidly developing and have extremely high market demands. The technology is deeply deposited in the field of Raman spectroscopy abroad, and the related technology of the Raman spectrometer is in the front of the world and occupies most of markets in the field of Raman spectrometers in China. In the aspect of handheld and portable raman spectrometers, foreign manufacturers such as must daycare (BWTEK), samer fly (Thermo Fisher Scientific), ocean Optics (Ocean Optics) and japan physics (Rigaku) have all put forward corresponding handheld/portable raman spectrometers, and are widely applied to fields such as college scientific research, security inspection security, food and drug safety, environmental protection and cultural relics protection. However, the raman spectrum has less application in medical clinic, because the biological fluid is more complex, the content of disease markers is lower, and the peak value of the raman spectrum is lower and cannot be detected.

And the SERS spectrum has good application prospect in body fluid analysis and disease diagnosis due to the special advantages of the SERS spectrum in a biological complex system. For example, jung et al encapsulate silver nanowires in a flexible substrate, trace detection of methamphetamine in sweat for real-time monitoring of physical condition; the silver nano square is self-assembled on a substrate by Ling et al of Nanyang, and the progesterone content in urine of a pregnant woman is detected to judge the abortion risk of the pregnant woman; tan et al label-free detection of bilirubin levels in blood by modifying graphene-silver nanoparticles on a paper substrate. However, most of commonly used surface enhanced raman substrates are hard substrates, so that sampling and detection are separated, the convenience of operation is still to be improved, and popularization and application of the technology are limited.

The integrated tear separation detection device comprises a substrate, and a silicon dioxide microsphere layer, a gold sputtering layer and a gold nanoparticle layer which are sequentially laminated on the surface of the substrate. The lower half part of the surface enhanced Raman spectrum detection substrate is an inactive space silicon dioxide array, can store dirt such as biological protein, and the surface of the surface enhanced Raman spectrum detection substrate is formed by nano particles and a gold sputtering layer to form a high-strength and high-density SERS active area, and can bind substances with strong interaction with gold.

According to the surface enhanced Raman spectrum detection substrate and the application thereof, disclosed by the patent document with the publication number of CN111896522B in the prior art, the first medium layer and the second medium layer are wrapped on the surface of the metal nano particle or the metal micro-nano structure on the substrate, so that the direct contact between the metal nano particle or the metal micro-nano structure and the external chemical environment can be effectively isolated, the Raman signal intensity of tear fluid can be obviously enhanced, and the effective detection of human eye infection sources can be realized by utilizing the surface enhanced Raman effect.

The prior art described above has improved the surface enhanced raman spectroscopy detection substrate for tear detection to some extent, but the prior art still requires conventional detection analysis. The conventional tear component detector is a commercial instrument based on liquid chromatography-mass spectrometry (HPLC-MS), and can accurately analyze the content of various disease markers in tear to diagnose diseases. However, the device is large and expensive, the operation is complex, the required tear sample content is large, the detection time is long, and the purpose of real-time diagnosis cannot be achieved. Therefore, there is a need to create a miniaturized, portable, popular, and intelligent integrated tear collection and detection machine that specifically recognizes disease markers in tears and makes early diagnosis of disease.

Furthermore, there are differences in one aspect due to understanding to those skilled in the art; on the other hand, as the inventors studied numerous documents and patents while the present invention was made, the text is not limited to details and contents of all that are listed, but it is by no means the present invention does not have these prior art features, the present invention has all the prior art features, and the applicant remains in the background art to which the rights of the related prior art are added.

Disclosure of Invention

Aiming at the defects that the Raman spectrum for tear detection is heavy, inconvenient to carry and incapable of quantitatively detecting diseases, which are proposed by the prior art, the application provides a tear detection system based on Raman scattering signals, which can be operated at any time and any place conveniently, and aims to civilize a mode of detecting corresponding diseases through tear, and can not only qualitatively detect diseases, but also quantitatively detect diseases.

The application provides a tear detection system, which is used for detecting and analyzing tear by using a surface enhanced Raman spectrum technology, and at least comprises a basal part used for collecting tear and a detection part used for analyzing the tear on the basal part, wherein the basal part is used for draining disease markers in the tear from a collection area to a specific strip according to a specific recognition mode, the detection part is used for carrying out qualitative and/or quantitative detection on diseases based on Raman scattering signal changes on the specific recognized strip, and a first light beam of the detection part excites a Raman scattering signal of the strip on the basal part and transmits the Raman scattering signal to an analysis module of the detection part in a second light beam mode.

According to the application, surface Enhanced Raman Spectroscopy (SERS) and detection test paper are combined, and quantitative trace detection can be carried out on dry eye markers and allergy markers at least in a short time under the condition of trace tear sample content. The close-packed nanoparticle film for testing is transferred to a hydrophilic area by a Langmuir-Blodgett film preparation method to form a multi-band SERS detection area. The SERS test paper with multiple detection areas can change the modification of the strip according to the requirement, and monitor multiple indexes in tear.

Preferably, the base portion comprises at least a first band capable of specifically recognizing a dry eye marker, the first band comprising a polypeptide sequence modified on the nanoparticle layer by means of a hydrophobic linkage.

Preferably, a nanoparticle layer with the polypeptide sequence is deposited on the colloidal reagent pad of the first strip, which is arranged on the substrate layer of the substrate portion, wherein the polypeptide sequence comprises a specific recognition segment capable of falling off upon contact with the dry eye marker, and the raman scattering signal of the nanoparticle layer of the first strip is changed accordingly.

Preferably, the polypeptide sequence of the first band is preferably designed as Cy3-Lys-Pro-Leu-Gly-Leu-Dap (Dnp) -Ala-Arg-Cy-SH, the specific recognition segment of the polypeptide sequence that is specifically recognized for matrix metalloproteinase-9 that is a marker for dry eye is preferably a Gly-Leu segment, and when the polypeptide sequence captures matrix metalloproteinase-9 or moves to the polypeptide sequence, the specific recognition segment of the polypeptide sequence-Gly-Leu segment-is separated from the polypeptide sequence, i.e., the Gly-Leu segment of the polypeptide sequence is sheared off, such that the polypeptide sequence on the first band is reduced in Raman scattering signal compared to the polypeptide sequence that is not sheared, and the greater the magnitude of the raman scattering signal is reduced in comparison to the collected tear fluid on the first band, the greater the amount of matrix metalloproteinase-9 in the unit tear that is the matrix metalloproteinase-9 is captured by the polypeptide sequence, and the more Raman scattering signal is attenuated in the first band. Through the above-mentioned scheme, the detection portion can accurately acquire the specific content of xerophthalmia marker on the first strip in unit tear, can qualitatively detect whether to suffer from xerophthalmia in addition to, can also quantitatively detect the severity of xerophthalmia.

Preferably, the base portion further comprises at least a second band capable of specifically recognizing the allergic marker, said second band comprising biotin modified on the nanoparticle layer by means of a hydrophobic linkage.

Preferably, the nanoparticle layer with the biotin is deposited on a colloidal reagent pad of the second strip, which is arranged on a substrate layer of the substrate portion, wherein the biotin is capable of reacting as an antibody with an allergic marker to capture the allergic marker, and the raman scattering signal of the nanoparticle layer of the second strip 140 is thus changed. The biotin on the second strip is designed as a thiol-labeled Cy 3-biotin-avidin, the thiol-labeled Cy 3-biotin-avidin is capable of specifically recognizing an allergic marker, immunoglobulin E (IgE), and the thiol-labeled Cy 3-biotin-avidin is capable of reacting as an antibody with an allergic marker IgE capable of acting as an antigen, thereby capturing the allergic marker IgE, so that the thiol-labeled Cy 3-biotin-avidin on the second strip is enhanced in raman scattering signal as compared with the thiol-labeled Cy 3-biotin-avidin which is not reacted with the allergic marker, and the enhancement amplitude is correlated with the immunoglobulin E as a Guo Ming marker in the collected tear fluid on the second strip, the higher the content of the immunoglobulin E per tear is, the more the number of the immunoglobulin E per tear fluid is, and the greater the enhancement amplitude of the raman scattering signal on the second strip is also. By the above scheme, the detection part can accurately acquire the specific content of the allergic marker on the second strip in the unit tear, and can quantitatively detect the severity of the allergic symptoms besides qualitatively detecting whether the allergic symptoms exist.

Preferably, the base portion further comprises at least a third strip for negative control, the third strip comprising a nanoparticle layer, the nanoparticle layer of the third strip being modified with a fluorescent label.

Preferably, the nanoparticles of the third strip are deposited on the colloidal reagent pad of the third strip, which is disposed on the substrate layer of the base portion, wherein the raman scattering signals of the nanoparticle layer of the third strip at the front and rear stages of contact with tear fluid are the same. Therefore, the third band, which does not change, is mainly used for negative control, excluding raman scattering signal changes caused by other light effects.

Preferably, the nanoparticle layers of the first, second and third strips are gold or silver nanoparticle layers. The nanoparticles may be selected from metal nanoparticles. The metal nano particles can be at least one of spheres, ellipsoids, cuboids, cubes and pyramids; the metal nano particles can be at least one of solid, hollow shell and multi-layer shell structures; the metal nano particles are at least one of single particles, double particles and agglomerated particles; the metal nano particles are at least one of gold, silver, aluminum and copper; the diameter of the metal nano particles is 1 nanometer to 10 micrometers.

Specifically, the nanoparticle layer adopts a gold or silver nanoparticle layer, and semiconductors and other metals such as copper and platinum also have certain SERS performance, but the enhancement degree of Raman signals of the gold or silver nanoparticle is far lower than that of the gold or silver nanoparticle.

Preferably, the basal part at least comprises a basal layer used for limiting the acquisition area and the strip, and the end part of the basal layer, which is attached to the skin of the human body, is in a micro arc shape. The utility model has the advantages that tears of human eyes mainly flow from the corners of eyes, and the contact ends of the micro arc shapes can collect tears more fully.

Preferably, the collection area is arranged on the surface of the micro-arc end part of the basal layer, and the edge of the collection area, which is close to the micro-arc of the basal layer, is designed into a micro-arc structure.

Preferably, a plurality of strips are attached to the substrate layer, a plurality of strips are connected with one side of the collection area opposite to the micro-arc structure, the first strips, the second strips and the third strips are arranged in parallel and are provided with intervals, the strips are subjected to hydrophilization treatment, and the intervals between the strips are subjected to hydrophobization treatment. The hydrophilic and hydrophobic treatment facilitates tear flow to the strips, and the parallel arrangement of the strips helps maintain consistent tear flow to the different strips.

Preferably, the strip on the base portion is capable of specifically recognizing one or more disease markers in tear fluid by altering the way the modification of the strip. Specifically, glucose in tear fluid may be used as a marker for diabetes, uric acid in tear fluid may be used as a marker for gout disease, and bilirubin in tear fluid may be used as a marker for jaundice disease. In this context, the term "specific recognition" means that different modifications on the strip can react specifically with different disease markers, and that the specific reaction means that, for a certain modification, the modification will bind to, react with, etc. only a certain disease marker, and thus the raman scattering signal on the strip will change.

The invention designs a first strip capable of detecting xerophthalmia and a second strip capable of detecting allergic diseases through tears, and a third strip for negative control is arranged between the first strip and the second strip, so that the detection of the xerophthalmia, allergic conjunctivitis and other diseases can be carried out once through collecting a trace amount of tears, and the allergic conjunctivitis and the xerophthalmia with similar symptoms at eyes can be judged differently, the problem that the existing tear detection test paper can only detect a specific one of the ocular diseases is solved, especially for patients possibly suffering from xerophthalmia and allergic conjunctivitis, as obvious symptoms do not exist in the early stage of the xerophthalmia, the symptoms of the allergic conjunctivitis are similar in the later stage of the xerophthalmia, the symptoms of the allergic conjunctivitis are determined after the detection only aiming at the allergic conjunctivitis in the early stage of the detection, and the possibility of the xerophthalmia is ignored, so that the dry eye is aggravated in many early stages of the patients, and the dry eye is caused by the fact that the dry eye is not detected and treated timely; even if the tear fluid is not ignored, tear fluid is required to be collected for multiple times and detected for multiple times, so that the consumption of medical resources is increased. The polypeptide sequences, the avidin and the fluorescent markers which are specially designed in the invention are respectively modified on the first strip, the second strip and the third strip, so that different signal change conditions can appear when different components in tear are detected, and patients suffering from xerophthalmia or allergic conjunctivitis can be effectively distinguished according to the specific signal change conditions, or two diseases suffer from the same time; further, the severity of the disease can be quantitatively determined by the specific numerical change of the signal on each band, thereby determining the dosage of the drug in the treatment regimen.

Drawings

FIG. 1 is a simplified overall structure schematic of a base portion of the present application;

FIG. 2 is a simplified top view schematic of the base portion of the present application;

fig. 3 is a schematic view of the light path direction structure of the detecting unit of the present application.

List of reference numerals

100: a base portion; 200: a detection unit; 110: a collection area; 120: a first strip; 130: a third strip; 140: a second strip; 150: a base layer; 151: a micro arc shape; 152: a hydrophobic region; 211: a first optical fiber; 212: a second optical fiber; 213: a detection port; 221: a first light beam; 222, a step of; a second light beam; 231: a first lens; 232: a second lens; 233: a third lens; 241: a narrow band pass filter; 242: a high-pass filter; 250: a reflecting mirror; 260: a dichroic mirror.

Detailed Description

The present application will be described in detail with reference to fig. 1 to 3.

Fig. 1 is a schematic diagram showing the overall structure of a base part of a tear detection system according to the present application, which uses a surface-enhanced raman spectroscopy to detect and analyze tear, the system comprising a base part 100 for collecting tear, and a detection part 200 for analyzing tear on the base part 100, wherein the base part 100 directs disease markers of tear from a collection area 110 to specific bands according to a specific recognition mode, and wherein a first light beam 221 of the detection part 200 excites raman scattering signals of different tear molecules on the base part 100 and transmits the signals to an analysis module of the detection part 200 in the form of a second light beam 222.

The conventional raman spectrum detection method is to transmit an excitation beam output by a laser to a sample to be detected through a raman probe so as to excite a raman optical signal, and then transmit the raman optical signal to a spectrometer for detection. The first beam 221 is an excitation beam in the raman spectroscopy field, and the second beam 222 is an excited raman optical signal.

Tears have a similar environment to blood and contain a large amount of water, proteins, lipids, cytokines, small molecule metabolites and the like, and the components are affected by ocular surface diseases and have content changes, so that certain ocular and body diseases can be detected and monitored through the changes of the components in tears. But detection of tear fluid is exceptionally difficult due to the small number of samples. The gold standard for detecting the component content of tear fluid is to detect by a liquid chromatograph-mass spectrometer, but the equipment is large and expensive, the operation is complex, and a small, portable and popular tear disease marker detection test paper needs to be created.

According to the application, surface Enhanced Raman Spectroscopy (SERS) and detection test paper are combined, and quantitative trace detection can be carried out on dry eye markers (MMP-9, matrix metalloproteinase-9) and allergy markers (IgE) in a short time (15 min) under the condition of trace tear sample content (2 mu L).

Preferably, as shown in fig. 2, the base portion 100 includes at least a first band 120 capable of specifically recognizing a dry eye marker, wherein the first band 120 includes a nanoparticle layer and a polypeptide sequence modified on the nanoparticle layer, and the dry eye marker is capable of disengaging a specific recognition segment in the polypeptide sequence when in contact with the polypeptide sequence.

Specifically, the polypeptide sequence of the first band 120 is preferably designed as Cy3-Lys-Pro-Leu-Gly-Leu-Dap (Dnp) -Ala-Arg-Cy-SH, the specific recognition segment of the polypeptide sequence that is specifically recognized for matrix metalloproteinase-9 that is a marker for dry eye is preferably a Gly-Leu segment, and when the polypeptide sequence captures matrix metalloproteinase-9 or moves to the polypeptide sequence, the specific recognition segment of the polypeptide sequence-Gly-Leu segment-is disengaged from the polypeptide sequence, i.e., the Gly-Leu segment of the polypeptide sequence is sheared off, such that the Raman scattering signal of the polypeptide sequence on the first band 120 is reduced compared to that of the polypeptide sequence that is not sheared, and the reduced amplitude is associated with matrix metalloproteinase-9 that is a marker for dry eye in the collected tear fluid on the first band 120, the greater number of matrix metalloproteinase-9 units in the tear is greater, and the more of the Gly-Leu segment in the polypeptide sequence on the first band 120 is sheared, the more Raman scattering signal of the first band is also reduced. By the above-described means, the detecting unit 200 can accurately obtain the specific content of the dry eye marker per unit tear on the first strip 120, and can quantitatively detect the severity of dry eye in addition to qualitatively detecting whether or not dry eye is present.

Preferably, as shown in fig. 2, the base part 100 further includes at least a second strip 140 capable of specifically recognizing the allergic marker, the second strip 140 includes a nanoparticle layer therein, and biotin modified on the nanoparticle layer, and the biotin is capable of reacting with an antigen-antibody of the allergic marker to capture the allergic marker.

Specifically, the biotin on the second strip 140 is designed as a thiol-labeled Cy 3-biotin-avidin, the thiol-labeled Cy 3-biotin-avidin is capable of specifically recognizing an allergic marker, immunoglobulin E (IgE), and the thiol-labeled Cy 3-biotin-avidin is capable of reacting as an antibody with the allergic marker IgE capable of acting as an antigen, thereby capturing the allergic marker IgE, so that the thiol-labeled Cy 3-biotin-avidin on the second strip 140 enhances raman scattering signal of the thiol-labeled Cy 3-biotin-avidin compared with that of the thiol-labeled Cy 3-biotin-avidin which does not react with the allergic marker, and the enhancement amplitude is related to the immunoglobulin E as an allergic marker in the collected tears on the second strip 140, the higher the content of the immunoglobulin E in the unit tears, the more the thiol-labeled Cy 3-biotin-avidin captures the immunoglobulin E on the second strip 140, and the larger the raman scattering signal on the second strip 140. By the above-described means, the detection unit 200 can accurately acquire the specific content of the allergic marker in the unit tear on the second strip 140, and can quantitatively detect the severity of the allergic symptoms in addition to qualitatively detecting whether the allergic symptoms are present.

Preferably, as shown in fig. 2, the base part 100 further includes at least a third strip 130 for negative control, the third strip 130 includes therein a nanoparticle layer and a fluorescent dye modified on the nanoparticle layer, and the raman scattering signal of the nanoparticle layer in the third strip 130 is the same in the front and rear stages of contact with tear fluid, in other words, the raman scattering signal of the nanoparticle layer in the third strip 130 does not significantly change in the front and rear stages of contact with tear fluid.

Preferably, as shown in fig. 2, the base portion 100 of the present application comprises at least a base layer 150, the base layer 150 being configured to define the relative positions of the acquisition region 110 and the first, second and third strips 120, 140, 130.

Preferably, substrate layer 150 may alternatively be configured in a regular rectangular shape, or other shape that facilitates acquisition of tear fluid. Since tears of the human eye normally flow down from the canthus of the eye, the end of the base layer 150 that contacts the human skin can be designed to conform to the micro-arc 151 of the eye shape.

Preferably, the collecting area 110 is disposed on the surface of the end of the micro arc 151 of the base layer 150, and the edge of the collecting area 110 near the micro arc 151 of the base layer 150 adopts a micro arc structure.

Preferably, a plurality of strips are attached to the substrate layer 150, the plurality of strips are connected to a side of the acquisition region 110 opposite to the micro-arc structure, and the first strip 120, the second strip 140, and the third strip 130 are disposed in parallel and spaced apart from each other.

Preferably, the spaces between the first, second, and third strips 120, 140, 130 are hydrophobized to form a plurality of hydrophobic regions 152. Specifically, wax printing may be applied to the spaced apart regions of the first, second, and third strips 120, 140, 130 on the substrate layer 150, and further, the regions of the substrate layer 150 not covered by the acquisition regions 110, 120, 140, 130 may be applied with wax printing such that the regions not covered by the acquisition regions 110, 120, 140, 130 are hydrophobic regions 152.

Preferably, the acquisition region 110 is made of absorbent cotton or other material capable of absorbing tear fluid to ensure adequate collection of tear fluid from the eye.

Preferably, the nanoparticle layers of the first, second and third strips 120, 140 and 130 are gold or silver nanoparticle layers. In general, when the distance between noble metal nanoparticles such as gold and silver is 10nm or less, localized plasmon resonance is generated, and a relatively strong raman scattering signal can be generated. In recent years, it has been found that semiconductors and other metals such as copper and platinum also have certain raman scattering properties, but far less enhanced than those of gold and silver nanoparticles. According to the application, a self-assembly-based method is adopted to carry out gas-liquid interface self-assembly on gold or silver nano particles, so that the nano particles form a close-packed monolayer film, the distance between the nano particles is ensured to be enough to generate stronger Raman scattering signals, and the uniformity of the Raman scattering signals of a substrate is also ensured.

Preferably, the application also provides a preparation method of the gold nanoparticle layer, and the preparation method of the nanoparticle layer should not be regarded as limiting the technical scheme of the application, so that the preparation of the nanoparticle layer belongs to conventional means for those skilled in the art. The application provides, by way of illustration only, a method for preparing a gold nanoparticle layer, comprising the following steps:

preparing a gold chloride solution with the mass percentage of 1%, a disodium tannate dihydrate solution with the mass percentage of 4%, a tannic acid solution with the mass percentage of 1%, a 50 millimole potassium carbonate solution, acetone and deionized water in advance;

1mL of chloroauric acid solution is added into 79mL of deionized water, and ultrasonic oscillation is carried out for 5 minutes;

then the solution is placed in a constant temperature oil bath pot at 60 ℃ for preheating for 5 minutes and vigorously stirred;

then adding 1mL of trisodium citrate dihydrate, 0.1mL of tannic acid and 0.15mL of potassium carbonate into 18.85mL of deionized water to prepare a reducing solution;

rapidly adding the reducing solution into preheated chloroauric acid solution, and vigorously stirring for 1h at the same temperature, wherein the solution is changed from light yellow to wine red;

after the reaction is finished, cooling the product at room temperature to obtain gold nanoparticle sol;

Adding 30mL of acetone into the gold nanoparticle sol, and centrifuging at 10000rpm for 10min to obtain a precipitate, wherein the precipitate is gold nanoparticles;

and depositing gold nano particles on the colloidal gold reagent pad to obtain a gold nano particle layer.

Preferably, the colloidal gold reagent pad of the first strip 120, the second strip 140 and the third strip 130 of the present application is hydrophilized by plasma treatment. In general, a person skilled in the art performs hydrophilization treatment using a plasma cleaning machine, and the principle of hydrophilization of the plasma cleaning machine is that active particles in plasma react with the surface of a material to form hydrophilic groups, so that the surface of the material has hydrophilicity.

Preferably, the substrate layer 150 itself has a certain flexibility, and a silicon wafer, a plastic sheet, or the like may be used, and a cellulose acetate film is coated on the surface of the substrate layer 150. The surface of the substrate layer 150 is generally composed of cellulose or glass fibers because they have little to no binding force to the biological sample. After tear fluid enters collection region 110, substrate layer 150 should be capable of transporting tear fluid in a smooth, continuous and uniform manner, and substrate layer 150 may serve to accelerate the release of analyte flow to other components of the strip, and may also be used to pre-treat tear fluid prior to its transport, which may include separation of sample components, removal of interference, adjustment of pH, etc.

Preferably, the strips on the base portion 100 of the present application are capable of achieving different marker detection by changing the modifications on the strips.

Each molecule has its own raman fingerprint spectrum, which is also why raman can detect labels in complex systems, but the raman signal of a molecule is usually very weak. When nanoparticles of noble metals such as gold and silver exist, and the gaps of the nanoparticles reach about 10nm, localized plasmon resonance (LSPR) is generated, so that surface-enhanced Raman scattering (SERS) is generated. The raman signal intensity of SERS is 4-5 orders of magnitude higher than ordinary raman signal intensity. In general, most SERS substrates are hard substrates in which metal nanoparticles are carried by photolithography, ion beam etching, or the like, and the substrates need to be subjected to raman test after pretreatment of body fluid and dripping onto the substrates during body fluid detection. According to the application, the paper-based SERS substrate is designed, the detection nanoparticles are carried on the Schirmer test paper in a simple self-assembly mode to form a plurality of strips, various disease markers in tear can be detected simultaneously after each strip is modified differently, the paper-based substrate can be directly put on an eyelid for tear collection, and real-time SERS signal output can be carried out on the strips. The raman results of disease markers in tears are compared and clearly correlated with disease marker components in tears and blood.

Preferably, the present application is also capable of detecting at least the levels of glucose, uric acid and bilirubin in tears by varying the modifications on the strips of the base portion 100, for diagnosing diabetes, gout and jaundice patients, respectively.

Preferably, the first light beam 221 of the detection unit 200 of the present application excites raman scattering signals of different tear molecules on the base unit 100 and is transmitted to the analysis module of the detection unit 200 in the form of the second light beam 222. Specifically, first light beam 221 is emitted by a laser connected to first optical fiber 211 and second light beam 222 is received by an analysis module connected to second optical fiber 212, which may employ a spectrometer, more specifically.

Example 2

The embodiment is improved and supplemented on the basis of embodiment 1, and repeated descriptions are omitted.

The present application proposes a tear detection system including a base portion 100 for collecting tear fluid and a detection portion 200 for analyzing and detecting tear fluid. Specifically, the detection unit 200 analyzes the molecular components in the tear fluid by using a raman spectroscopic analysis technique. In the past, a large-scale Raman desktop is required for Raman detection, and in recent years, a handheld Raman instrument is used in the fields of explosion prevention, drug detection and the like, but qualitative results are output, and the handheld Raman instrument is less in application in biological fluid detection. On the basis of the work of the part, a handheld Raman instrument is used for designing an integrated platform for acquiring and detecting tears in real time, and semi-quantitative or even quantitative results are output through Raman signal intensity. SERS detection test paper is placed under eyelid to absorb tear, the detection area is soaked and then placed on a detection platform, and the result can be read out on a display screen within 10 seconds.

Preferably, as shown in fig. 3, the detecting portion 200 of the present application includes at least two optical paths, including an incident optical path and a reflected optical path, where the incident optical path and the reflected optical path are at least partially overlapped, a first light beam 221 in the incident optical path is emitted from the detecting port 213 of the detecting portion 200, and a second light beam 222 in the reflected optical path is emitted into the detecting portion 200 from the detecting port 213.

Preferably, as shown in fig. 3, the first light beam 221 of the incident light path enters from the first optical fiber 211 of the incident light path and enters the subsequent light path through the first lens 231, specifically, the first lens 231 is a collimating lens for collimating the first light beam 221 emitted from the laser at the first optical fiber 211. Collimation refers to the fact that light rays emitted from a single point are scattered and can become parallel light rays after being refracted at a certain angle, and collimation refers to the fact that scattered light rays are refracted into parallel light rays.

Preferably, as shown in fig. 3, the first light beam 221 in the incident light path passes through the narrow band-pass filter 241 in the form of light after passing through the first lens 231, and the narrow band-pass filter 241 is used for filtering the interference signal in the first light beam 221.

Preferably, as shown in fig. 3, the first light beam 221 in the incident light path passes through the narrow band-pass filter 241 and reaches the mirror 250, the mirror 250 reflects the first light beam 221 to the dichroic mirror 260, the dichroic mirror 260 is parallel to the mirror surface of the mirror 250, and the dichroic mirror 260 can reflect the first light beam 221 again. Specifically, the direction of the light beam reflected by the first light beam 221 through the reflecting mirror 250 is perpendicular to the direction of the light beam collimated by the first light beam 221, and then the direction of the light beam reflected by the dichroic mirror 260 is parallel to the direction of the light beam collimated by the first light beam 221.

Preferably, as shown in fig. 3, the first light beam 221 in the incident light path is reflected by the dichroic mirror 260, focused by the second lens 232, and emitted from the detection port 213 of the detection portion 200 onto a plurality of strips of the base portion 100. The second lens 232 is essentially a focusing collimating lens, and for the first light beam 221, the first light beam 221 enters from the convex surface of the second lens 232 and exits from the plane, thereby focusing the first light beam 221; as for the second light beam 222, the second light beam 222 reflected by the strip of the base portion 100 enters from the plane of the second lens 232 in a scattered state, and exits from the convex surface, and the second lens 232 collimates the second light beam 222.

Preferably, as shown in fig. 3, the second light beam 222 in the reflected light path is incident from the second lens 232 and collimated by the second lens 232, and the scattered light reflected by the strip is converted into collimated parallel light, and the second light beam 222 passes through the dichroic mirror 260 in the direction of the collimated light. The dichroic mirror 260 is characterized by being almost completely transparent to light of a certain wavelength and almost completely reflective to light of other wavelengths. For first beam 221 and second beam 222, first beam 221 is emitted by a laser, parameters may be set manually; the second light beam 222 is a raman light signal reflected by the baseband and has a wavelength that is significantly different from that of the first light beam 221, while the dichroic mirror 260 is capable of reflecting the first light beam 221 based on the difference between the two light beams, while the second light beam 222 is completely transmitted.

Preferably, as shown in fig. 3, the second light beam 222 passes through the dichroic mirror 260 and then the raman spurious signals in the second light beam 222 are filtered out by the high-pass filter 242.

Preferably, as shown in fig. 3, the second light beam 222 passes through the high pass filter 242, is focused by the third lens 233 and is collected by the second optical fiber 212, and the second optical fiber 212 transmits the raman scattered signal in the second light beam 222 to the analysis module.

Preferably, as shown in fig. 3, the first optical fiber 211 of the detecting part 200 is connected to a laser, and a first light beam 221 emitted from the laser is emitted from the first optical fiber 211. Specifically, as one of the core devices, a laser needs to be considered in terms of stability, output power, laser intensity, sensitivity to temperature, fluorescence of a sample, and the like. Because the target is a portable detection platform, a relatively small-sized laser is required.

More specifically, the center wavelength of the laser light emitted from the laser of the detection portion 200 is: 785 nm.+ -. 0.5nm; the wavelength stability of the laser emitted by the laser is as follows: (+ -0.005 nm@8h; the linewidth of the laser emitted by the laser is as follows: <0.1nm; the continuous output power of the laser is: 0-500mW; the processing system of the laser is sized as follows: 76.2mm by 63.5mm by 18mm; the configuration parameters of the first optical fiber 211 of the laser connection are at least: 105 μm and 0.22NA; the light interface of the laser adopts an SMA905 interface.

Preferably, the second optical fiber 212 of the detection part 200 receives the raman scattering signal (second light beam 222) reflected by the base part 100 and transmits it to an analysis module, specifically, the analysis module is directly connected to the second optical fiber 212, and more specifically, the analysis module may be provided as a spectrometer. The spectrometer is used as a light splitting instrument, and can image incident light rays on different positions of an image plane according to different wavelengths, so that light can be dispersed in space. Currently, a micro spectrometer is commonly adopted as a light splitting system in a handheld/portable raman spectrometer. The micro spectrometer is the "heart" of the handheld/portable raman spectrometer, and the performance of the micro spectrometer directly affects the spectral resolution, sensitivity and spectral detection range of the raman spectrometer.

More specifically, due to portability requirements, the spectrometer of the detection section 200 is sized to: 91mm by 60mm by 34.5mm; the wavelength range of the light which can be analyzed by the spectrometer is 750nm-1100nm; the optical resolution of the spectrometer is: 0.35-1nm; the detector of the spectrometer is designed as follows: 2048 linear array CMOS; the integration time of the spectrometer is 0.5ms-10s; the full spectrum signal to noise ratio of the spectrometer is: 600:1.

preferably, another relatively important component in the detecting section 200 is a filter. In order to avoid the side band spectral components or stray light of the laser from entering the excitation light path and to prevent interference caused by raman signals of the incident optical fiber itself, a narrow bandpass filter 241 is required to be provided in the excitation light path. The raman signal is weak and has an intensity of about 10 of the excitation light intensity ^-6 To 10 ^-8 The excitation light needs to be strong enough to obtain an observable raman signal. At the same time of improving the energy of the excitation light, the Rayleigh scattered light of the substance and the reflected light intensity of the sample surface are also enhanced, which can lead to the difficult recognition of the Raman scattered light signal and even the saturation of the signal read by a spectrometer, so thatThe spectral signal quality is degraded. Therefore, suppression or filtering of Rayleigh scattered light is a fundamental requirement of Raman spectroscopy.

More specifically, the size of the narrowband pass filter 241 of the detection section 200 is:tolerance + -0.1 mm, wherein +.>An outer diameter of the narrow band-pass filter 241 is 6mm, and 1mm indicates a wall thickness of the narrow band-pass filter 241 is 1mm; the center wavelength of the narrow bandpass filter 241 is 784.5mm + -1.5 mm; the narrow bandpass filter 241 has a full width at half maximum (FWHM) of: 6nm; the transmittance of the narrow band pass filter 241 is: more than or equal to 93 percent abs@785nm; the stopband of the narrow bandpass filter 241 is:

≥OD5Abs，OD6Avg585-776.5nm，

or alternatively, the process may be performed,

≥OD5Abs，OD6Avg792.5-1150nm。

it should be noted that the above-described embodiments are exemplary, and that a person skilled in the art, in light of the present disclosure, may devise various solutions that fall within the scope of the present disclosure and fall within the scope of the present disclosure. It should be understood by those skilled in the art that the present description and drawings are illustrative and not limiting to the claims. The scope of the invention is defined by the claims and their equivalents.

Claims

1. A substrate part based on a specific recognition modification is characterized by comprising at least

A first band (120) capable of specifically recognizing a dry eye marker;

a second band (140) capable of specific recognition for an allergic marker;

wherein, the liquid crystal display device comprises a liquid crystal display device,

the first band (120) comprises a polypeptide sequence modified on the nanoparticle layer;

the second strip (140) comprises biotin modified on the nanoparticle layer.

2. The substrate portion based on a specific recognition modification according to claim 1, wherein the specific recognition modification comprises at least a polypeptide sequence capable of specifically recognizing a dry eye marker and biotin capable of specifically recognizing an allergic marker,

the polypeptide sequence is designed to be Cy3-Lys-Pro-Leu-Gly-Leu-Dap-Ala-Arg-Cys-SH, or Cy3-Lys-Pro-Leu-Gly-Leu-Dap-Ala-Arg-Cys-SH, and the specific recognition segment Gly-Leu of the polypeptide sequence can be separated from the polypeptide sequence after the specific recognition segment Gly-Leu contacts with the xerophthalmia marker;

the biotin is designed as sulfhydryl-labeled Cy 3-biotin-avidin, which is capable of reacting as an antibody with an antigen-antibody of an allergic marker to capture the allergic marker.

3. Tear detection system for detection and analysis of tears using surface-enhanced raman spectroscopy, characterized in that it comprises at least a base portion (100) according to claim 1;

and a detection unit (200) for analyzing tear fluid on the base unit (100);

the base part (100) drains the disease marker in tear from the collecting area (110) to a specific strip according to a specific recognition mode, the detection part (200) carries out qualitative and/or quantitative detection on the disease based on the Raman scattering signal change of the strip after the specific recognition,

wherein a first light beam (221) of the detection section (200) excites a raman scattering signal of a strip on the substrate section (100) and is transmitted in the form of a second light beam (222) to an analysis module of the detection section (200).

4. A tear detection system according to claim 3, characterized in that the nanoparticle layer with a polypeptide sequence capable of specifically recognizing a dry eye marker is deposited on a colloidal reagent pad of a first strip (120), the colloidal reagent pad of the first strip (120) being arranged on a substrate layer (150) of the substrate portion (100),

wherein the polypeptide sequence includes a specific recognition segment capable of shedding upon contact with a dry eye marker, and wherein the raman scattering signal of the nanoparticle layer of the first band (120) is altered.

5. The tear detection system according to claim 3 or 4, characterized in that the nanoparticle layer with biotin capable of specifically recognizing an allergic marker is deposited on a colloidal reagent pad of a second strip (140), the colloidal reagent pad of the second strip (140) being arranged on a base layer (150) of the base portion (100),

wherein the biotin is capable of reacting as an antibody with an antigen of the allergic marker to capture the allergic marker, and the raman scattering signal of the nanoparticle layer of the second strip (140) is changed accordingly.

6. The tear detection system according to any one of claims 3 to 5, characterized in that the base portion (100) further comprises at least a third strip (130) for a negative control, the third strip (130) comprising a nanoparticle layer, the nanoparticle layer of the third strip (130) being modified by a fluorescent label.

7. The tear detection system according to any one of claims 3 to 6, characterized in that the nanoparticles of the third strip (130) are deposited on a colloidal reagent pad of the third strip (130), the colloidal reagent pad of the third strip (130) being arranged on a substrate layer (150) of the substrate portion (100),

Wherein the nanoparticle layer of the third strip (130) has the same raman scattering signal at the front and rear stages of contact with tear fluid.

8. The tear detection system according to any one of claims 3 to 7, characterized in that the first strip (120), the second strip (140), the third strip (130) are arranged in parallel and are provided with a space therebetween, a plurality of strips are subjected to hydrophilization treatment, and a plurality of spaces between strips are subjected to hydrophobization treatment.

9. The tear detection system according to any one of claims 3 to 8, characterized in that the nanoparticle layers of the first strip (120), the second strip (140) and the third strip (130) are gold or silver nanoparticle layers.

10. The tear detection system according to any one of claims 3 to 9, characterized in that a number of the strips on the base part (100) are capable of specifically identifying one or more disease markers in tear fluid corresponding to a modification by changing the modification of the strips.