CN115236054A - Diagnosis device based on surface enhanced Raman scattering - Google Patents
Diagnosis device based on surface enhanced Raman scattering Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The present disclosure provides a surface enhanced raman scattering-based diagnostic device, comprising: an inlet module for receiving a liquid to be analysed; the reaction module is provided with a receiving hole in a first area, an output hole in a second area and communicated with the receiving hole through a flow channel, at least one chemical reagent group is arranged in the flow channel, the reaction module receives the liquid to be analyzed conveyed by the inlet module through the receiving hole, the liquid to be analyzed can flow through the chemical reagent group in the flow channel to obtain the liquid to be analyzed carrying the nano particles, and the liquid to be analyzed carrying the nano particles can flow to the second area of the reaction module; the detection module can receive the liquid to be analyzed carrying the nano particles and conveyed by the output hole of the reaction module; the laser can irradiate the liquid to be analyzed carrying the nanoparticles and received by the detection module through the output hole of the reaction module, and the liquid to be analyzed is excited to generate surface enhanced Raman scattering.
Description
Technical Field
The present disclosure belongs to the field of detection technology, and particularly relates to a diagnostic device based on surface enhanced raman scattering.
Background
In 2019, 2.29 billion human malaria cases and 409000 related deaths were estimated to occur globally (world health organization, 2020), and early malaria diagnosis was crucial in order to reduce morbidity and mortality (Ashley et al, 2018).
The "gold standard" for malaria diagnosis is microscopic examination of Giemsa-stained blood smears (Ashley et al, 2018), but this method is time consuming and requires skilled personnel to identify infections, such as a detection limit of 4-20 parasites/μ L blood.
Although researchers have developed various malaria diagnostic techniques (Ragavan et al, 2018), each of these techniques also has its own drawbacks. For example, rapid diagnostic test methods (RDTs) have been developed that enable rapid diagnosis, but these rapid diagnostic methods have low detection sensitivity (Ashley et al, 2018), high probability of false positive or false negative results (Reboud et al, 2019), and no quantitative parasite detection (Rifaie-Graham et al, 2019) to assess the progression of malaria disease. On the other hand, most sensitive detection methods capable of quantitative detection (e.g., enzyme linked immunosorbent assays, real-time quantitative polymerase chain reaction) require a laboratory environment for handling (Ashley et al, 2018 reboud et al, 2019 rifaie-Graham et al, 2019. Among these diagnostic techniques, raman spectroscopy, as an optical method, is capable of providing a vibrational fingerprint for the molecular species under examination, has the potential to diagnose malaria rapidly, and does not require skilled operators, but it has insufficient sensitivity, making it difficult to identify low levels of parasitomias at an early stage of diagnosis (Patel et al, 2019).
Surface Enhanced Raman Scattering (SERS) has been used to improve the detection sensitivity and signal intensity of raman signals of hemozoin, a biological crystal, a unique biomarker of malaria infection (Garrett et al, 2015 wang et al, wood et al, 2011; yuen and Liu, 2013).
In SERS strategies, SERS-active nanoparticles need to be brought close to hemozoin for raman signal amplification, and most SERS studies mix the existing nanoparticles with hemozoin pigment obtained from lysed blood prior to SERS measurement to achieve this requirement. Since the complete blood lysis method also frequently lyses the parasite, this lysis step releases and disperses highly localized aggregated hemozoin nanocrystals from each vacuole, forming multiple disintegrated nanocrystals within a larger surrounding volume, which may require further concentration and extraction (Wang et al, 2020). In addition, the average distance between the nanoparticles and the sparsely distributed hemozoin crystals is increased, which results in a lower level of SERS signal.
In contrast, we (Chen et al, 2016 b) synthesized SERS nanoparticles in the parasite to enhance raman signals from hemozoin in malaria-infected blood. The advantage of forming nanoparticles within the parasite is that the SERS signal is greatly improved due to the close proximity between the nanoparticles and the hemozoin and/or its high concentration vacuoles within the parasite. This close proximity is difficult to achieve with other existing nanoparticles or nanostructures (Garrett et al, 2015 laing et al, 2017 perez-Guaita et al, 2018 wang et al, 2020), because these existing nanoparticles are not small enough to penetrate multiple membrane barriers, such as the parasite plasma membrane and the parasite vacuolar membrane, and approach the internal hemozoin.
Disclosure of Invention
In order to solve at least one of the above technical problems, the present disclosure provides a surface enhanced raman scattering-based diagnostic apparatus, comprising:
an inlet module for receiving a liquid to be analyzed;
a reaction module, wherein a first region of the reaction module is provided with a receiving hole, a second region of the reaction module is provided with an output hole, the receiving hole is communicated with the output hole through a flow channel, at least one chemical reagent group is arranged in the flow channel, the reaction module receives the liquid to be analyzed conveyed by the inlet module through the receiving hole, the liquid to be analyzed can flow through the chemical reagent group in the flow channel to obtain the liquid to be analyzed carrying nanoparticles, and the liquid to be analyzed carrying nanoparticles can flow to the second region of the reaction module;
a detection module capable of receiving a liquid to be analyzed carrying nanoparticles conveyed via the output aperture of the reaction module;
the laser can irradiate the liquid to be analyzed carrying the nanoparticles and received by the detection module through the output hole of the reaction module, and excite the liquid to be analyzed carrying the nanoparticles to generate surface enhanced Raman scattering.
According to the diagnosis device based on the surface enhanced Raman scattering of the at least one embodiment of the present disclosure, the chemical reagent set comprises a plurality of chemical reagents, each chemical reagent is arranged in the flow channel in a manner of a dry chemical reagent point, and each dry chemical reagent point is arranged at intervals in sequence.
According to the diagnostic device based on the surface enhanced raman scattering of at least one embodiment of the present disclosure, the number of the chemical reagent groups is 3 or 4, and each chemical reagent group is sequentially arranged at intervals.
According to at least one embodiment of the present disclosure, the surface-enhanced raman scattering-based diagnostic device has an aperture size of the receiving aperture larger than an aperture size of the output aperture.
According to at least one embodiment of the present disclosure, the reaction module includes a first substrate and a first sealing film stack layer disposed on the first substrate, and a through groove is formed in the first sealing film stack layer to form the flow channel.
According to at least one embodiment of the present disclosure, the entrance module and the detection module are disposed on the same side of the reaction module.
According to the diagnosis device based on the surface enhanced Raman scattering of at least one embodiment of the present disclosure, the detection module includes two single-layer sealing films, a glass fiber filter paper and an aluminum foil, the glass fiber filter paper is sandwiched between the two single-layer sealing films and is commonly disposed on the aluminum foil, each single-layer sealing film has a circular hole thereon, and the circular holes of the two single-layer sealing films are aligned; through the round holes on the two single-layer sealing films, the nano particles or the nano particles and the markers in the liquid to be analyzed, which is conveyed by the output hole of the reaction module, can be deposited on the glass fiber filter paper, and the filtered liquid is discharged along the pin holes of the aluminum foil.
According to the diagnosis device based on the surface enhanced raman scattering of at least one embodiment of the present disclosure, the aperture of the circular holes on the two single-layer sealing films of the detection module is equal to and larger than the aperture of the output hole.
According to the diagnosis device based on the surface enhanced Raman scattering of at least one embodiment of the present disclosure, the aluminum foil is provided with a pinhole, and the pinhole on the aluminum foil is not aligned with the round holes on the two single-layer sealing films of the detection module.
According to at least one embodiment of the present disclosure, the size of the glass fiber filter paper is smaller than that of the single layer sealing film of the detection module.
The surface-enhanced Raman scattering-based diagnostic apparatus according to at least one embodiment of the present disclosure further includes a filtering module disposed between the inlet module and the first region of the reaction module to filter the liquid to be analyzed conveyed by the inlet module.
According to at least one embodiment of the present disclosure, the surface-enhanced raman scattering-based diagnostic apparatus includes a single-layer sealing film, a second sealing film stack layer, and a first-stage filter paper sandwiched between the single-layer sealing film and the second sealing film stack layer, the second sealing film stack layer is in close contact with the reaction module, the single-layer sealing film and the second sealing film stack layer are both provided with a circular hole, and the circular hole on the single-layer sealing film is aligned with the circular hole on the second sealing film stack layer.
According to the diagnostic device based on the surface enhanced raman scattering of at least one embodiment of the present disclosure, the second sealing film stack layer is formed by stacking more than four single-layer sealing films.
According to the diagnostic device based on the surface enhanced raman scattering of at least one embodiment of the present disclosure, the first sealing film stack layer is formed by stacking four or more single-layer sealing films.
According to at least one embodiment of the present disclosure, the size of the primary filter paper is smaller than the size of the single-layer sealing film of the filter module and smaller than the size of the second sealing film stack of the filter module.
According to at least one embodiment of the present disclosure, the inlet module includes a cap portion, a third sealing film stack layer and a second substrate, the cap portion is disposed on the third sealing film stack layer, the third sealing film stack layer is disposed on the second substrate, the second substrate is in close contact with the single-layer sealing film of the filter module, the inlet module has a liquid channel, and a liquid to be analyzed can enter the reaction module through the liquid channel and the filter module.
According to at least one embodiment of the present disclosure, the cover portion has a cover through hole formed therein, the third sealing film stack layer has a circular hole formed therein, the second substrate has a pinhole formed therein, and the liquid passage of the inlet module is formed by the cover through hole of the cover portion, the circular hole of the third sealing film stack layer, and the pinhole of the second substrate.
According to the diagnostic device based on surface enhanced raman scattering of at least one embodiment of the present disclosure, the third sealing film stack layer is formed by stacking eight or more single sealing films.
A surface enhanced raman scattering based diagnostic device according to at least one embodiment of the present disclosure is used for diagnosis of malaria in blood.
According to at least one embodiment of the present disclosure, the surface-enhanced raman scattering-based diagnostic device includes silver nanoparticles.
The surface-enhanced Raman scattering-based diagnostic device according to at least one embodiment of the present disclosure further includes a housing, and the assembled reaction module, the filtration module, the inlet module, and the detection module are disposed within the housing.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the disclosure and together with the description serve to explain the principles of the disclosure.
Fig. 1 is a schematic diagram of a surface enhanced raman scattering based diagnostic device/chip and photographs of various components of one embodiment of the present disclosure. FIG. 1 (a) is an exploded schematic view and the component parts of the device/chip; wherein (I) a reaction module having 3sets of dry chemical points a, B, C, and D; (II) a filtration module; (III) an inlet module; (IV) a detection module; (b) preferred detailed dimensions of the reaction module; (c) - (f) are photographs of the sub-modules corresponding respectively to the modules (I-IV); (g) A photograph of the entire assembled SERS device/chip taken in the laboratory; (h) photographs of manual syringe pumps made by the present disclosure. In fig. 1, a: silver nitrate; b: sodium hydroxide; c: hydroxylamine Hydrochloride (hydrochlorane Hydrochloride); d: sodium chloride; FG filter, i.e. glass fiber filter paper; g1 filter is primary filter paper; according to a preferred embodiment of the present disclosure, all the holes on the device/chip of the present disclosure are 3mm in diameter, unless noted as pinholes (pinhole diameter is preferably 0.81 mm), or labeled as 5mm in diameter. A pinhole on the aluminum foil of the detection module is not aligned with the laser path, while another pinhole on the transparent sheet of the reaction module is used to pass the laser for SERS measurements.
In fig. 2, (a) - (c) are concentrations of 10 obtained for the optimized diagnostic device/chip of the present disclosure -5 M, and (d) - (f) are the blood-infected SERS spectra obtained from the optimized diagnostic device/chip of the present disclosure with a parasitemia level of 0.05%, as compared to other devices/chips:
(a) 1X, 4X, 6X, 8X, 10X theoretical mass (m) theo ) (deposited chemical species), and (d) 1 ×,6 ×,8 ×,10 ×,12 × theoretical mass (m) theo ) (deposited chemicals); (b) 0mM,1.2mM, 2.4mM,3.6mM,6mM (sodium chloride), and (e) 04mM,1.6mM,2.4mM,3.2mM, 4.8mM (sodium chloride); (c) 1 chemical group, 2 chemical groups, 3 chemical groups, 4 chemical groups, and (f) 1 chemical group, 2 chemical groups, 3 chemical groups, 4 chemical groups, 5 chemical groups (each chemical group has 4 dry chemical dots). The "× 10" in the figure represents the intensity of the corresponding spectrum multiplied by 10 for visualization.
In fig. 3, (a) is a control of the SERS spectrum of the plasmodium-infected blood and the SERS spectrum of the normal blood, and the concentrations of the hemozoin are: 2X 10 -8 M，4×10 -8 M，9×10 -8 M，1.8×10 -7 M，2.7×10 -7 M，4.5×10 -7 M，9.0×10 -7 M，1.8×10 -6 M and 4.5X 10 -6 And M. Each average spectrum (black) is the average of 25 spectra (grey) of 5 different samples, each sample having 5 random positions. (b) Is a correlation between an estimated hemozoin concentration and a reference hemozoin concentration, wherein the estimated hemozoin concentration is obtained by PLS-LOO technique from SERS spectra obtained using a hemozoin optimized device/chip of the present disclosure.
Fig. 4 to 11 are enlarged views of (a) to (h) in fig. 1, respectively.
Detailed Description
The present disclosure will be described in further detail with reference to the drawings and embodiments. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limitations of the present disclosure. It should be further noted that, for the convenience of description, only the portions relevant to the present disclosure are shown in the drawings.
It should be noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. Technical solutions of the present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
Unless otherwise indicated, the illustrated exemplary embodiments/examples are to be understood as providing exemplary features of various details of some ways in which the technical concepts of the present disclosure may be practiced. Accordingly, unless otherwise indicated, features of the various embodiments may be additionally combined, separated, interchanged, and/or rearranged without departing from the technical concept of the present disclosure.
The use of cross-hatching and/or shading in the drawings is generally used to clarify the boundaries between adjacent components. As such, unless otherwise specified, the presence or absence of cross-hatching or shading does not convey or indicate any preference or requirement for a particular material, material property, size, proportion, commonality between the illustrated components and/or any other characteristic, attribute, property, etc., of a component. Further, in the drawings, the size and relative sizes of components may be exaggerated for clarity and/or descriptive purposes. While example embodiments may be practiced differently, the specific process sequence may be performed in an order different than that described. For example, two processes described consecutively may be performed substantially simultaneously or in an order reverse to that described. In addition, like reference numerals denote like parts.
When an element is referred to as being "on" or "over," "connected to" or "coupled to" another element, it can be directly on, connected or coupled to the other element or intervening elements may be present. However, when an element is referred to as being "directly on," "directly connected to" or "directly coupled to" another element, there are no intervening elements present. To this end, the term "connected" may refer to physically, electrically, etc., and may or may not have intermediate components.
For descriptive purposes, the present disclosure may use spatially relative terms such as "under 8230; \8230;,"' under 8230; \8230; below 8230; under 8230; above, on, above 8230; higher "and" side (e.g., in "side wall)", etc., to describe the relationship of one component to another (other) component as shown in the figures. Spatially relative terms are intended to encompass different orientations of the device in use, operation, and/or manufacture in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "at" \8230; "below" may encompass both an orientation of "above" and "below". Further, the devices may be otherwise positioned (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, when the terms "comprises" and/or "comprising" and variations thereof are used in this specification, the presence of stated features, integers, steps, operations, elements, components and/or groups thereof are stated but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof. It is also noted that, as used herein, the terms "substantially," "about," and other similar terms are used as terms of approximation and not as terms of degree, and as such, are used to interpret inherent deviations in the measured, calculated, and/or provided values that would be recognized by one of ordinary skill in the art.
The surface enhanced raman scattering based diagnostic device/chip of the present disclosure will be described in detail below with reference to fig. 1 to 11.
Materials and methods
Chemical substances and materials:
silver nitrate (AgNO 3), rhodamine 6G (R6G), sodium chloride (NaCl), percoll, sorbitol and sodium hydroxide (NaOH), these chemicals granules/powders being purchased from mox, germany (Merck, germany).
RPMI 1640 medium and
from ThermoFisher, USA. Hydrochloric acid hydroxyAmines (HONH 2. HCl) were purchased from MP Biomedicals, USA. Volumes of Parafilm PM 996M, first grade filter paper, and glass microfiber filters (GF/B grade) were purchased from Whatman, inc., UK. Clear film (PP 2500) was purchased from 3M company, usa. A paper cutter (Silhouette Cameo 4) was purchased from Silhouette, inc. of USA.
Ethical statement
Whole blood was donated by healthy non-malaria immune adult volunteers at singapore national university hospital. Informed written consent was obtained from all donors according to protocols approved by the institutional review board of southern university of singapore (IRB-2018-02-031).
Plasmodium falciparum parasite culture
The P.falciparum strain 3D7 was cultured in fresh red blood cells, supplemented with 5% albumin and 3% O2, 5% CO2, the remainder being N2 gas at 4% hematocrit in RPMI 1640 medium and cultured at 37 ℃ as described above (Trager and Jensen, 1976). Growth medium was replaced daily with fresh whole RPMI.
Smears were made on microscope slides to check for parasitemia. Late schizont parasites were purified using 70% Percoll centrifugation (Kutner et al, 1985). To synchronize more closely, the purified schizont stage parasites are allowed to re-invade fresh red blood cells. After 5-6 hours of growth, the parasites were treated with 5% sorbitol to remove all late parasites (Aley et al, 1986) and 96-99% of the parasites were in the ring phase. Blood samples were stored at 4 ℃, and all measurements were completed within about 30 days after parasite culture was completed.
A surface enhanced raman scattering based diagnostic device according to an embodiment of the present disclosure, with reference to fig. 1 and 4, includes:
an inlet module for receiving a liquid to be analyzed;
the system comprises a reaction module, a first area of the reaction module is provided with a receiving hole, a second area of the reaction module is provided with an output hole (the output hole can be a pinhole capable of receiving laser light), the receiving hole is communicated with the output hole through a flow channel, at least one chemical reagent set (set) is configured in the flow channel, the reaction module receives liquid to be analyzed conveyed by an inlet module through the receiving hole, the liquid to be analyzed can flow through the chemical reagent set (set) in the flow channel to obtain liquid to be analyzed carrying nano particles, and the liquid to be analyzed carrying the nano particles can flow to the second area of the reaction module;
the detection module can receive the liquid to be analyzed carrying the nano particles, which is conveyed through the output hole of the reaction module;
the laser can irradiate the liquid to be analyzed carrying the nanoparticles and received by the detection module through the output hole of the reaction module, and excite the liquid to be analyzed to generate Surface Enhanced Raman Scattering (Surface Enhanced Raman Scattering).
The nanoparticles described in the present disclosure are preferably silver nanoparticles, and may of course be gold nanoparticles, etc., and those skilled in the art can select/adjust the kind of nanoparticles within the scope of the present disclosure.
The technical solution of the present disclosure is illustrated below with an example of silver nanoparticles as nanoparticles.
The liquid to be analyzed described in the present disclosure may be human blood or animal blood, and the human blood or animal blood may be conveyed to the reaction module of the diagnostic apparatus through the inlet module, the reaction module of the present disclosure has a flow channel, a chemical reagent set (set) configured in the flow channel may be dissolved by the human blood or animal blood, the dissolved chemical reagent set performs a chemical reaction to generate silver nanoparticles, and the silver nanoparticles may exist in the form of silver nanosol. Thus, the human blood or the animal blood to be analyzed is mixed with the silver nanoparticles.
The method comprises the steps of irradiating the liquid to be analyzed carrying silver nanoparticles by using laser to generate surface enhanced Raman scattering, and acquiring a spectrum of the surface enhanced Raman scattering to perform spectral analysis, so that whether the liquid to be analyzed contains a biomarker related to a specific disease (such as malaria) or not can be diagnosed, and rapid field diagnosis of the specific disease can be realized.
Preferably, the chemical reagent set (set) of the surface enhanced raman scattering-based diagnostic device of the present disclosure includes a plurality of chemical reagents, each of which is disposed in the flow channel in the form of a dried chemical reagent dot, and each of the dried chemical reagent dots is sequentially disposed at intervals.
The chemical reagent set (set) of the present disclosure may include silver nitrate, sodium hydroxide, hydroxylamine hydrochloride, and sodium chloride, that is, four chemical reagents, the four chemical reagents are sequentially disposed in the flow channel at intervals, and the intervals between the chemical reagents may be equal.
According to a preferred embodiment of the present disclosure, the number of chemical reagent groups of the surface enhanced raman scattering-based diagnostic apparatus of the present disclosure is 3 or 4, and each chemical reagent group is sequentially disposed at intervals.
According to the experiment (described in detail below) of the present disclosure, when the number of the chemical reagent sets is 3sets or 4sets, the better surface enhanced raman scattering can be generated, and those skilled in the art can adjust the number of the chemical reagent sets, the types of the chemical reagents included in the chemical reagent sets, and the like, all falling within the protection scope of the present disclosure.
The aperture size of the receiving hole of the reaction module of the surface enhanced raman scattering-based diagnostic device of the present disclosure is preferably larger than the aperture size of the output hole.
The present disclosure enables the dry chemical reagent dots to be sufficiently dissolved by the analyte-to-be-analyzed liquid by setting the aperture size of the receiving hole to be larger than that of the output hole.
Referring to fig. 1 and 5, the reaction module of the surface-enhanced raman scattering-based diagnostic device of the present disclosure preferably includes a first substrate and a first sealing film stack layer disposed on the first substrate, and the through-grooves are formed on the first sealing film stack layer to form the flow channels described above.
The "substrate" described in the present disclosure may be a sheet-shaped substrate made of various materials, such as a sheet-shaped substrate made of PVC (Polyvinyl chloride), and is preferably a transparent substrate. Those skilled in the art can select/adjust the material, type, etc. of the "substrate" in light of the disclosure, and all such materials and types are within the scope of the disclosure.
According to one embodiment of the present disclosure, the receiving hole is disposed on the first substrate and is in communication with the first end of the through-slot, and the output hole is disposed on the first substrate and is in communication with the second end of the through-slot.
According to a preferred embodiment of the present disclosure, referring to fig. 1 and 5, both the first end of the through groove and the second end of the through groove are circular or elliptical in shape.
The reaction module of the diagnostic device of the present disclosure further includes a third substrate, the first stack of sealing films being sandwiched between the first substrate and the third substrate.
As shown in fig. 1, the entrance module and the detection module of the surface enhanced raman scattering-based diagnostic device of the present disclosure are disposed on the same side of the reaction module.
Referring to fig. 1 and 9, the detection module of the surface enhanced raman scattering-based diagnostic device of the present disclosure preferably includes two single-layer sealing films, a glass fiber filter paper and an aluminum foil, the glass fiber filter paper is sandwiched between the two single-layer sealing films and is commonly disposed on the aluminum foil, each single-layer sealing film has a circular hole thereon, and the circular holes of the two single-layer sealing films are aligned; the nano particles or nano particles and the markers (and the filter residues in the liquid to be analyzed) of the liquid to be analyzed carrying the silver nano particles and conveyed by the output hole of the reaction module through the round holes on the two single-layer sealing films can be deposited on the glass fiber filter paper, and the filtered liquid is discharged along the pinhole of the aluminum foil
Referring to fig. 1, the diameters of the circular holes on the two single-layer sealing films of the detection module are equal to each other and are larger than the diameter of the output hole.
Referring to fig. 1 and 9, a pin hole (needle hole) is preferably formed in the aluminum foil of the test module, and the pin hole is not aligned with the circular holes of the two single-layer sealing films of the test module.
Preferably, the size of the glass fiber filter paper of the detection module is smaller than that of the single-layer sealing film of the detection module.
By setting the size of the glass fiber filter paper of the detection module of the diagnostic device disclosed by the disclosure to be smaller than that of the single-layer sealing film, the glass fiber filter paper can be completely arranged between the two single-layer sealing films.
For the diagnosis device based on the surface enhanced raman scattering of each of the above embodiments, it is preferable that the diagnosis device further includes a filtering module disposed between the inlet module and the first region of the reaction module to filter the liquid to be analyzed conveyed by the inlet module.
According to a preferred embodiment of the present disclosure, the filter module of the surface-enhanced raman scattering-based diagnostic apparatus of the present disclosure includes a single-layer sealing film, a second sealing film stack layer, and a first-stage filter paper (G1 filter) sandwiched between the single-layer sealing film and the second sealing film stack layer, the second sealing film stack layer is in close contact with the reaction module, the single-layer sealing film and the second sealing film stack layer both have a circular hole, and the circular hole on the single-layer sealing film is aligned with the circular hole on the second sealing film stack layer.
The second sealing film stacking layer of the filtering module is formed by stacking more than four layers of single-layer sealing films.
The first sealing film stack layer of the reaction module of the diagnostic device is formed by stacking more than four single-layer sealing films.
The adjustment/selection of the number of the single-layer sealing films of the second sealing film stack layer of the filtration module and the adjustment/selection of the number of the single-layer sealing films of the first sealing film stack layer of the reaction module, which are taught by the technical solutions of the present disclosure, all fall within the protection scope of the present disclosure.
According to a preferred embodiment of the present disclosure, referring to fig. 1, the size of the primary filter paper of the filter module is smaller than the size of the single layer of the sealing film of the filter module and smaller than the size of the second sealing film stack of the filter module.
Referring to fig. 1 and 4, the inlet module of the surface-enhanced raman scattering-based diagnostic apparatus of the present disclosure includes a cover portion, a third sealing film stack layer, and a second substrate, the cover portion is disposed on the third sealing film stack layer, the third sealing film stack layer is disposed on the second substrate, the second substrate is in close contact with the single-layer sealing film of the filtration module, the inlet module has a liquid passage, and a liquid to be analyzed can enter the reaction module through the liquid passage and the filtration module.
Referring to fig. 1, 4 and 8, a cover through hole is formed on the cover portion of the inlet module, a circular hole is formed on the third sealing film stack layer, a pin hole is formed on the second substrate, and a liquid passage of the inlet module is formed by the cover through hole of the cover portion, the circular hole of the third sealing film stack layer and the pin hole of the second substrate.
Wherein the third sealing film stacking layer of the inlet module is formed by stacking more than eight single-layer sealing films. It is within the scope of the present disclosure for a person skilled in the art to adjust/select the number of the single sealing films of the third sealing film stack layer of the inlet module in light of the teachings of the present disclosure.
For the surface enhanced raman scattering-based diagnostic device of each of the above embodiments, it is preferable that a housing (not shown) is further included, and the assembled reaction module, filtering module, inlet module and detection module are disposed in the housing.
According to one embodiment of the present disclosure, the surface enhanced raman scattering-based diagnostic device/chip of the present disclosure is prepared by the following steps:
(1) The prepared material (filter paper, vial cap (visual cap), aluminum foil, sealing film, transparent film) was first patterned into a desired shape and size using a paper cutter, as shown in fig. 1 (a) and fig. 1 (b), with a pore diameter of preferably 3mm, unless otherwise specified in fig. 1.
(2) Manufacturing each submodule:
reaction module (please refer to fig. 1): the present disclosure stacks four pieces of sealing film (parafilm) together to form a first stacked layer of sealing film, wherein the present disclosure forms two holes (preferably 3mm in diameter) on each piece of sealing film, the two holes are communicated through a channel, i.e., a reaction module, the first stacked layer of sealing film is disposed on a first substrate (transducer slide) having a first hole and a second hole, the first hole is preferably a hole with a diameter of 3mm, the second hole is preferably a 21G pin hole, the first hole is aligned with one of the two holes of the first stacked layer of sealing film, and the second hole is aligned with the other of the two holes of the first stacked layer of sealing film, as shown in fig. 1 (b) and fig. 1 (c).
Filter module (please refer to fig. 1): four pieces of sealing film are stacked together to form a second sealing film stack layer, and a layer of primary filter paper is sandwiched between the second sealing film stack layer and a single layer of sealing film layer, thereby forming a filter module. Wherein, a hole is formed on the second sealing film stacking layer, a hole is also formed on the single-layer sealing film, and the hole on the second sealing film stacking layer is aligned with the hole on the single-layer sealing film.
Inlet module (please refer to fig. 1): a hole, preferably 3mm in diameter, is punched in the vial cap (visual cap), which may be a vial cap cut from a vial. The vial cap is disposed on a third lidding film stack layer (refer to (e) in fig. 1), wherein the third lidding film stack layer is disposed on a second substrate (transducer slide), wherein the third lidding film stack layer is formed of an eight-piece lidding film (parafilm) stack.
Detection module (please refer to fig. 1): a piece of glass fiber filter paper (preferably 7mm × 7mm in size, refer to (f) in fig. 1) was sandwiched between two single-layer sealing films, each having circular holes aligned with each other, preferably 5mm in diameter, and placed on an aluminum foil (Al foil) having a pinhole (Needle hole) not aligned with the circular holes of the two single-layer sealing films, i.e., the pinhole was offset from the central axis of the circular holes of the two single-layer sealing films. The misaligned pinhole serves to release the pressure built up by the analyte at the detection module during pumping and minimizes the expulsion of nanoparticles and parasites.
Another piece of aluminum foil (a sacrificial sheet of Al foil) was used to cover the channels described above, this piece of aluminum foil temporarily replaced the substrate (the covering transparency slide) shown in fig. 1 (a), the third substrate, and the assembled device/chip was heated to 120 ℃ (possibly on a hot plate for 4 minutes).
It is noted that during heating, another piece of aluminum foil as described above is in contact with the surface of the hot plate. Due to the high temperature, no chemicals are placed in the channels described above during the heating process.
After the heating process is complete, the other piece of aluminum foil described above is removed and chemicals are dropped into the channel described above.
AgNO 3 (20.5. Mu.g) was dropped at a position 8mm from the edge of the channel, followed by sequentially dropping NaOH (14.4. Mu.g) and HONH 2 HCl (12.5 μ g), naCl (2.8 μ g), the spacing between the four chemicals is 4mm, see (a) in FIG. 1 and (B) in FIG. 1, i.e. the positions A, B, C, D in (a) in FIG. 1.
Next, these dropped chemicals were air-dried in a dark environment (the se chemicals power air dried in a dark environment). Sealing is performed to cover the third substrate, i.e., the substrate for covering, on top, as shown in fig. 1 (a), and then heated to 60 ℃ (which may be heated on a hot plate for 4 minutes) to adhere the first sealing film stack to the substrate for covering (transparency slide), the first sealing film stack being sealed between the first substrate and the third substrate. This heating temperature is low enough to prevent any unwanted chemical reaction of the reaction module prior to use, as shown in fig. 1 (g). Since many chemical reactions require higher temperatures, e.g. AgNO 3 Thermal decomposition reaction occurs at 160 ℃.
SERS measurement preparation-operation of device/chip
In each test, 20 μ l of test analyte was input to the inlet module of the device/chip for pumping. The present disclosure plugs a vial cap into a barrel flange of a syringe pump (see (h) in fig. 1), where the syringe pump is a self-made syringe pump of the present disclosure and the test analyte is driven to dry AgNO 3 The first spot in (a) of fig. 1, point a, is tested for analyte pair dryness before being driven to the next chemical spot (point B in (a) of fig. 1, point B)Dried AgNO 3 The chemical dots were dissolved for two minutes. The progress of the test analyte to the next dry chemistry spot is visually monitored by the operator through the third substrate, and may be monitored secondarily by markings on the syringe of the injection pump. This procedure is also applicable to the processing of the remaining dry chemistry spots (third spot, i.e., C to twelfth spot in FIG. 1 (a)), and finally pushing the test analyte into the detection module.
The third substrate and the first sealing film stack layer were peeled off prior to SERS measurement.
The laser (fig. 1 (a), dashed arrow) was focused through a pinhole on the first substrate onto a glass fiber filter paper of the detection module for SERS measurement.
At a concentration of 10 -5 R6G of M and blood of parasitemia levels 0.05% of malaria-infected blood were used to find the best chemical configuration for SERS measurements of R6G and hemozoin.
In the SERS measurement of R6G, 20 μ l of an aqueous solution of R6G was used as the analyte for the test. In a malaria-related SERS measurement, 10 μ l of deionized water was mixed with 10 μ l of normal blood or malaria-infected blood, where various parasitemia levels (ring phase) were 0%, 0.0025%, 0.005%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, and 0.5%, corresponding to 0M, 2 × 10%, respectively -8 M、4×10 -8 M、9×10 -8 M、1.8×10 -7 M、2.7×10 -7 M、4.5×10 -7 M、9.0×10 -7 M、1.8×10 -6 M and 4.5X 10 -6 And M, which are used as the analytes for the test in turn. Suppose that human blood contains 5X 10/ml 9 Individual Red Blood Cells (RBC), the plasmodium pigment concentration in the ring phase is 0.22pg/RBC, and the molecular weight of the plasmodium pigment is 1229g/mol (Chen et al.2016b; yuen and Liu, 2012).
Raman instrument and data processing
A micro-raman system (innoRam-785s,b &w tek,us) was coupled with a video microscope (BAC 151A, B & W TEK, US) in a back scattering system to evaluate the characteristics of the raman instrument.
In SERS measurementIn the measurement, a laser having a wavelength of 785nm (unless otherwise stated, the laser power was 5mW at the time of blood measurement and 1mW at the time of R6G measurement) was focused on a sample through an objective lens (60x, N.A. 0.85) of a video microscope. After the sample is excited by laser, the Raman signal emitted by the sample is collected by the same objective lens and has the spectral resolution of 4cm -1 The grating of (a) diffracts for detection. Each spectrum was acquired with an exposure time of 20 seconds and accumulated 4 times. To obtain the final spectra, each raw spectrum is subjected to a five-point moving average process to remove noise before removing the fluorescent background (Zhang et al, 2009). The spectra shown in the results section are the average of the final spectra obtained from 5 different samples, each sample having 5 random positions (unless otherwise indicated). In addition, SERS spectra of malaria-infected blood was from 1535cm -1 To 1645cm -1 Modelling as a superposition of two vibration characteristics, i.e. C α C m (1586 cm in blood -1 ) V and v C＝C (hemozoin 1624cm in hemozoin) -1 ) ) are superimposed. To calculate the Raman signal contribution (v) from only Plasmodium pigments C＝C At 1624cm -1 ) The present disclosure will use this segment (from 1622cm -1 To 1626cm -1 ) The fit is the sum of two lorentzian functions and the area under the fitted lorentzian curve is calculated.
Results
Optimization of device/chip surface enhanced Raman spectroscopy performance by different chemical components
Fig. 2 shows the SERS performance of two different sets of devices/chips that were optimized for SERS measurements for R6G ((a) to (c) in fig. 2) and infected blood ((d) to (f) in fig. 2) using different amounts of chemicals.
Changes in SERS spectra of R6G and hemozoin of different amounts of AgNO3, naOH and HONH 2. HCl deposited on dry chemical spots were evaluated in fig. 2 (a) and fig. 2 (d), respectively. According to the method of Leopold and Lendl (2003), the theoretical mass of 1mM AgNO3 is 3.4. Mu.g, and the theoretical mass of 3mM NaOH (m) theo ) 2.4. Mu.g, 1.5mMThe theoretical mass of HONH 2. HCl is 3.4. Mu.g. Mass as theoretical mass (m) was deposited separately on the diagnostic device/chip of the present disclosure theo ) Fold different amounts of chemicals for SERS measurement of R6G and hemozoin. SERS results showed a 6 Xm deposition in R6G analytes theo Of 8 x m in a hemozoin analyte theo The device/chip has the best SERS enhancement effect. As shown in fig. 2 (b) and (e), the effect of different NaCl concentrations in the device/chip was investigated for SERS experiments with R6G and hemozoin, respectively. The SERS intensity of R6G and hemozoin (b) and (e) in fig. 2) was best at 2.4mM NaCl concentration. Fig. 2 (e) and (f) show SERS spectra of R6G and hemozoin analytes, respectively, with R6G and hemozoin flowing through different groups of four dried chemical spots, respectively. The results showed that the dried chemicals of group 3 ((c) in fig. 2) and group 4 ((f) in fig. 2) had the highest R6G SERS intensity and hemozoin SERS intensity, respectively. We observed that an R6G optimized device/chip with 6 times the theoretical mass of chemical deposition, 2.4mM NaCl, and 3sets (3 sets) of dry chemicals provided the best R6G SERS signal. We also note that the hemozoin optimized device/chip with 8 times the theoretical mass of chemical deposited, 2.4mM NaCl, and 4sets (4 sets) of dry chemicals produced the highest hemozoin SERS signal.
Quantitative diagnosis of hemozoin concentration in malaria-infected blood
Fig. 3 (a) shows SERS spectra obtained using a hemozoin optimization device/chip for an uninfected blood and a malaria-infected (ring stage) blood parasitemia levels of 0.0025% to 0.5%.
At 951cm -1 (ν 3 CH 3 )、1003cm -1 (ν 47 )、1087cm -1 (ν C＝C )、1247cm -1 (Amide III ), 1345cm -1 (C 2vinyl H)、1375cm -1 (ν 4 )、1448cm -1 (δ，CH 2 /CH 3 ) And 1584 cm -1 (δ，C α C m ) There is a clear SERS Raman peak (in FIG. 3 (a))
). This is similar to the vibration signatures reported in other literature (Atkins et al, 2017 chen et al, 2016a chen et al, 2016 b), which are found in both malaria-infected blood and normal blood. In addition, 1053cm was observed in blood infected with malaria -1 And 1624cm -1 (ν C＝C ) The prominent peak at (t, in (a) in figure 3) is comparable to the SERS result obtained using the laboratory SERS method (Chen et al, 2016 b). In FIG. 3 (b) is according to 1624cm -1 SERS peaks, estimated concentration plotted using PLS-loov technique (RMSEP 0.3 μm) versus reference concentration of hemozoin. The present disclosure also found that according to a series of t-tests (t-test, by mixing 1624cm -1 The peak at (A) is compared with the peak of a normal blood sample, P<0.05 At 20 nM) cyosomal or hemozoin concentrations, the lowest detectable parasitemia level was 0.0025%.
Discussion of the related Art
The present disclosure improves the SERS performance of the device/chip by optimizing the amount of chemicals used (fig. 2). First, by increasing the mass of the same proportion of chemical to compensate for incomplete dissolution of the chemical in the device/chip, the highest SERS signal occurs at 6 × m theo (FIG. 2 (a)) mass sum of 8 Xm theo (fig. 2 (d)) a mass of a deposition chemistry. In fact, the present disclosure contemplates that this ratio may vary for different types of chemicals, and may vary for the same type of chemical at different drying points (e.g., agNO) 3 Points 1, 5 and 9) are different, but in the study of the present disclosure, the present disclosure assumes that all of these are the same. Secondly, naCl was introduced into the device/chip ((b) and (e) in fig. 2) to induce Ag nanoparticle aggregation and form nanogap to further enhance SERS signal, which should correspond to other SERS structures reported in literature andsimilar (Han et al, 2011; min et al, 2018). These geometries (geogels) allow for more efficient SERS activity compared to silver nanoparticles, which are relatively sparsely distributed. Third, analytes flowing through three chemical groups ((c) in fig. 2) and four chemical groups ((f) in fig. 2) gave the best improvement, probably due to the enlargement of silver nanoparticle size as the analytes flow through more sets of dry chemicals, similar to the strategy used to grow other types of enlarged silver structures (Yuen and Liu, 2013). Larger diameter silver nanoparticles exhibit higher extinction cross-section values in the near infrared wavelength (Yu et al, 2017 yuen and Liu, 2013), resulting in higher SERS strength. After obtaining the optimal number of chemical groups, the decrease in SERS intensity of silver nanoparticles may be due to the further increase in size of silver nanoparticles, the decrease in nanoparticle density per hemozoin (hemozoin). Thus, the present disclosure enhances the SERS performance of two differently configured devices/chips based on two different test molecules: R6G in water ((a) to (c) in fig. 2) and hemozoin in blood ((d) to (f) in fig. 2). These results demonstrate the feasibility of on-site in-situ synthesis of SERS-active nanoparticles on a device/chip for sensitive in-situ measurements of chemical and biological analyte molecules.
Further, the optimized device/chip is used for representing the SERS performance of the R6G molecules. Compared with a spontaneous Raman signal, the R6G optimized device/chip effectively enhances the SERS signal of the R6G. Analytical Enhancement Factor (AEF) for the presently disclosed device/chip and Ag nanoparticles (ranging from 4 x 10) produced using other similar methods in a laboratory environment -3 To 7X 10 -5 ) Is obtained by
Et al, 2008; ju et al, 2017; leopold and Lendl, 2003), and comparable to other types of SERS devices/chips that form nanoparticles in situ (Zhao et al, 2016).
Furthermore, when using the PLS-loov technique, the Root Mean Square Error (RMSEP) of the predicted value (RMSEP) is 49 μm, and the SERS intensity has a good correlation with the R6G concentration, which is comparable to other SERS devices/chips that have been reported (Yaghobian et al, 2011). Thus, the present disclosure enables sensitive chemical SERS measurements, eliminating shelf-life issues in other types of SERS substrates due to the instant synthesis property. The methods of the present disclosure have also been demonstrated to enable SERS testing of biomolecules (e.g., hemozoin).
The present disclosure also detects the concentration of hemozoin in malaria-infected blood using the surface-enhanced raman scattering-based malaria diagnostic device/chip of the present disclosure (fig. 3). Fig. 3 (a) shows the SERS spectra of malaria-infected blood and malaria-uninfected blood obtained from the device/chip after optimization of hemozoin of the present disclosure. We note that the vibrational characteristics (e.g., v) derived from hemozoin biochemicals (hemozoin biochemicals) C＝C ) More prominent in SERS measurements of the present disclosure than other studies (Chen et al, 2016 a). Notably, in order to limit the considerable number of malaria biomarkers hemozoin to local high concentrations in the parasite and/or its vacuoles, only small amounts of water are used to lyse the blood on the device/chip. The stronger SERS signal of hemozoin may be due to AgNO 3 Aqueous solutions and other chemicals diffuse through multiple membrane barriers, which allows the formation of nanoparticles closer to the hemozoin nanocrystals that are concentrated in the vacuole, resulting in additional SERS enhancement. In contrast, other groups reported that without lysis, SERS spectral characteristics of membranes or other blood components predominated (with less contribution of hemozoin biomarkers) (Chen et al, 2016 a). Otherwise, a separate step is required to cleave all membranes to expose the hemozoin crystals (Garrett et al, 2015), which may result in the aggregated hemozoin nanocrystals being released within the vacuole and dispersed to a larger volume after cleavage, thereby reducing their local concentration and thus reducing the SERS signal. Thus, the strategy of the present disclosure promises to enable potential on-site malaria diagnosis by analyzing SERS spectra without the need for a laboratory environment and complex procedures (e.g., a step of lysing blood). FIG. 3 (b) evaluated the 1622cm previously described -1 To 1626cm -1 Fitting the area under the Lorentz curve to investigate the unique vibrational mode (v) contributed by hemozoin biocrytal C＝C ) For quantifying parasitemia levels to assess the progression of malaria disease. The RMSEP value in the results of the present disclosure (fig. 3 (b)) was 0.3 μm, superior to other types of SERS devices/chips reported in the literature for bacterial detection (RMSEP was 4 μm) (Morelli et al, 2018). AEF is not discussed in this disclosure because even at high parasitemia levels, hemozoin is difficult to detect inside the parasite without silver nanoparticles. Furthermore, if the cells and parasites are completely lysed to expose the hemozoin nanocrystals, the situation will become different compared to the experiments of the present disclosure (which are most likely to have unlysed parasites and/or their vacuoles). A detection limit of 0.0025% corresponds to 125 ring stages of parasite/μ l (assuming that the human normal blood contains 5X 10 9 One erythrocyte/ml) or a detection limit corresponding to 42 parasites/μ l when detecting schizogenic parasites. This estimate is based on the fact that the hemozoin concentration of the parasite at the schizont stage is approximately three times the hemozoin concentration at the ring stage (Serebrennikova et al, 2010). However, this detection limit is different from our previous work in a laboratory environment (Chen et al, 2016 b) (0.00005%). Differences in detection limits can be attributed to several potential factors. First, the dried chemical may not be completely dissolved in the blood sample due to insufficient mixing. Furthermore, the Raman system of the present disclosure for reading SERS devices/chips (innoRam-785s, b) compares to the Renishaw system (inVia, renishaw, UK) in our previous work (Chen et al, 2016 b)&W TEK, US) is a more compact and cost-effective Raman system, however, at the expense of reduced sensitivity. Another important factor affecting sensitivity is that the excitation wavelength in this disclosure is 785nm, which is different from 633nm, which is the excitation wavelength used in our previous work. For the same size of silver nanoparticles, the extinction cross-section calculation based on Mie light scattering of individual silver nanoparticles, an extinction coefficient at 633nm excitation is expected to be about 3 times higher than that at 785nm excitation (Abajo,2019; yu et al, 2017), therefore, theoretically, the detection limit could be increased 3-fold (about 14 schizont parasites/. Mu.l).
The instant synthesis of near analytes and SERS nanoparticles within the devices/chips of the present disclosure improves shelf life compared to other SERS schemes, and greatly reduces the required sample preparation procedures, e.g., no centrifuge is required to concentrate hemozoin, similar to RDTs. However, the disclosed device/chip can achieve higher sensitivity compared to the common RDT technique and has the advantage of quantifying hemozoin to indicate the severity and progression of malaria disease in patients. Furthermore, the diagnostic device/chip of the present disclosure neither requires a rigorous laboratory environment nor expensive equipment (e.g., clean room conditions and facilities) for diagnostic device/chip handling and manufacturing, and the technical solution of the present disclosure reduces the use and manufacturing costs of the diagnostic device/chip compared to most laboratory-based technologies. Therefore, further development of the disclosed technology and further development of low cost spectrometers can be aimed at creating a more efficient and highly sensitive SERS-based malaria diagnostic technique.
Conclusion
In conclusion, the diagnostic device/chip of the present disclosure is used for sensitive measurement of hemozoin in malaria field diagnosis, and the detection limit can reach 0.0025% parasitemia level, namely 125 parasites in ring body stage/μ l, and the detection limit can be improved by three times by simply adjusting the laser wavelength. The device/chip of the present disclosure can be operated in the field without a laboratory environment and can minimize the handling of hazardous chemical precursors. Notably, the devices/chips of the present disclosure can be easily manufactured and mass produced at low cost. More importantly, SERS-active nanoparticles can be synthesized instantaneously within the device/chip if hemozoin is present in the blood, which enables the formation of near-analyte nanoparticles, enabling stronger SERS signals and longer shelf life. Therefore, the technical scheme of the present disclosure enables SERS-based malaria diagnosis to be closer to large-scale, low-cost field detection.
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In the description herein, reference to the description of the terms "one embodiment/mode," "some embodiments/modes," "example," "specific example" or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment/mode or example is included in at least one embodiment/mode or example of the present disclosure. In this specification, the schematic representations of the terms used above are not necessarily intended to be the same embodiment/mode or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments/modes or examples. Furthermore, the various embodiments/modes or examples and features of the various embodiments/modes or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present disclosure, "plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
It will be understood by those skilled in the art that the foregoing embodiments are provided merely for clarity of disclosure and are not intended to limit the scope of the disclosure. Other variations or modifications may occur to those skilled in the art, based on the foregoing disclosure, and are still within the scope of the present disclosure.
Claims (10)
1. A surface enhanced raman scattering based diagnostic device, comprising:
an inlet module for receiving a liquid to be analyzed;
a reaction module, wherein a first region of the reaction module is provided with a receiving hole, a second region of the reaction module is provided with an output hole, the receiving hole is communicated with the output hole through a flow channel, at least one chemical reagent group is arranged in the flow channel, the reaction module receives the liquid to be analyzed conveyed by the inlet module through the receiving hole, the liquid to be analyzed can flow through the chemical reagent group in the flow channel to obtain the liquid to be analyzed carrying nano particles, and the liquid to be analyzed carrying nano particles can flow to the second region of the reaction module; and
a detection module capable of receiving a liquid to be analyzed carrying nanoparticles conveyed via the output aperture of the reaction module;
the laser can irradiate the liquid to be analyzed carrying the nanoparticles and received by the detection module through the output hole of the reaction module, and the liquid to be analyzed carrying the nanoparticles is excited to generate surface enhanced Raman scattering.
2. The surface-enhanced raman scattering-based diagnostic device of claim 1, wherein the set of chemical reagents comprises a plurality of chemical reagents, each chemical reagent being disposed in a dry chemical reagent spot within the flow channel, and each dry chemical reagent spot being disposed at sequential intervals.
3. The surface-enhanced raman scattering-based diagnostic device according to claim 2, wherein the number of the chemical reagent groups is 3 or 4, and each chemical reagent group is sequentially disposed at intervals.
4. The surface-enhanced raman scattering-based diagnostic device of claim 1, wherein an aperture size of said receiving aperture is larger than an aperture size of said output aperture.
5. The surface-enhanced raman scattering-based diagnostic device according to claim 1, wherein the reaction module comprises a first substrate and a first sealing film stack layer disposed on the first substrate, and the flow channel is formed by forming a through groove on the first sealing film stack layer.
6. The surface-enhanced Raman scattering based diagnostic device of any one of claims 1-5, wherein the inlet module and the detection module are disposed on the same side of the reaction module.
7. The surface-enhanced Raman scattering-based diagnostic device according to any one of claims 1 to 5, wherein the detection module comprises two single-layer sealing films, a glass fiber filter paper and an aluminum foil, wherein the glass fiber filter paper is sandwiched between the two single-layer sealing films and is commonly arranged on the aluminum foil, each single-layer sealing film is provided with a circular hole, and the circular holes of the two single-layer sealing films are aligned; through the round holes on the two single-layer sealing films, the nano particles or the nano particles and the markers in the liquid to be analyzed, which is conveyed by the output hole of the reaction module, can be deposited on the glass fiber filter paper, and the filtered liquid is discharged along the pinhole of the aluminum foil.
8. The surface-enhanced raman scattering-based diagnostic device of claim 7, wherein the diameter of the circular holes on the two single-layer sealing films of the detection module is equal to and larger than the diameter of the output hole.
9. The surface-enhanced raman scattering-based diagnostic device of claim 7 wherein said pinhole of said aluminum foil is not aligned with a circular hole in two single-layer sealing films of said detection module.
10. The surface-enhanced raman scattering-based diagnostic device of claim 7, wherein the size of the glass fiber filter paper is smaller than the size of the single layer sealing film of the detection module;
preferably, the device further comprises a filtering module, wherein the filtering module is arranged between the inlet module and the first area of the reaction module so as to filter the liquid to be analyzed conveyed by the inlet module;
preferably, the filtering module comprises a single-layer sealing film, a second sealing film stacking layer and primary filter paper clamped between the single-layer sealing film and the second sealing film stacking layer, the second sealing film stacking layer is in close contact with the reaction module, the single-layer sealing film and the second sealing film stacking layer are both provided with a round hole, and the round hole on the single-layer sealing film is aligned with the round hole on the second sealing film stacking layer;
preferably, the second sealing film stacking layer is formed by stacking more than four layers of single-layer sealing films;
preferably, the first sealing film stacking layer is formed by stacking more than four single-layer sealing films;
preferably, the size of the primary filter paper is smaller than that of a single layer of sealing film of the filter module and smaller than that of a second sealing film stacked layer of the filter module;
preferably, the inlet module comprises a lid portion, a third membrane stack layer and a second substrate, the lid portion is disposed on the third membrane stack layer, the third membrane stack layer is disposed on the second substrate, the second substrate is in close contact with the single-layer membrane seal of the filter module, the inlet module has a liquid passage through which the liquid to be analyzed can enter the reaction module through the filter module;
preferably, the lid portion is formed with a lid through hole, the third cover film stack layer is formed with a circular hole, the second substrate is formed with a pinhole, and the liquid passage of the inlet module is formed by the lid through hole of the lid portion, the circular hole of the third cover film stack layer, and the pinhole of the second substrate;
preferably, the third sealing film stack layer is formed by stacking more than eight single sealing films;
preferably, the diagnostic device is for diagnosis of malaria in blood;
preferably, the nanoparticles are silver nanoparticles;
preferably, the device further comprises a shell, and the assembled reaction module, the filter module, the inlet module and the detection module are arranged in the shell.
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