Magnetic bead enrichment SERS (Surface Enhanced Raman Scattering) probe sensing platform based on lung cancer marker protein-DNA (Deoxyribonucleic Acid) signal conversion as well as preparation method and application thereof

Abstract

The invention discloses a magnetic bead enrichment SERS (Surface Enhanced Raman Scattering) probe sensing platform based on lung cancer marker protein-DNA (Deoxyribose Nucleic Acid) signal conversion as well as a construction method and application of the magnetic bead enrichment SERS probe sensing platform. The target protein aptamer is an aptamer specifically bound with a carcino-embryonic antigen (CEA) protein; the SERS probe comprises gold nanoparticles, and the surfaces of the gold nanoparticles are covalently connected with a Raman reporter molecule sequence and capture DNA (Deoxyribose Nucleic Acid); the SERS probe is hybridized with the target protein aptamer on the first magnetic carrier compound by capturing DNA to form a DNA double-chain structure; the second magnetic carrier compound comprises a second magnetic carrier fixed with enriched DNA, and the enriched DNA sequence and the captured DNA are completely complementary. According to the invention, ultrasensitive and high-specificity quantitative detection of lung cancer marker protein (such as CEA) is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioanalytical chemistry and nanotechnology, and specifically relates to a magnetic bead enrichment SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion. BACKGROUND

[0002] Lung cancer is one of the highest incidence and mortality rates of malignant tumors worldwide, and its early diagnosis is crucial to improve patient survival. Carcinoembryonic antigen (CEA) as a broad-spectrum tumor marker, its concentration level in body fluids is closely related to the occurrence and development of lung cancer, therefore, developing a method that can quickly, sensitively and specifically detect CEA has important clinical value.

[0003] Surface-enhanced Raman spectroscopy (SERS) technology has great potential in the field of biological sensing due to its unique "fingerprint" recognition ability and theoretical detection sensitivity at the single molecule level. However, traditional SERS detection strategies often face two major challenges when directly applied to complex biological samples such as blood: first, the fluorescence and Raman background signals from the biological matrix interfere seriously and mask the target signal; second, the problem of false positives caused by non-specific adsorption, which together limit the actual application sensitivity and reliability in trace marker detection.

[0004] Chinese invention patent application CN202010540034.5 proposes a SERS sensor based on tetrahedral DNA structure, which can simultaneously detect multiple markers such as proteins and miRNAs on a single chip using rigid DNA scaffolds, achieving "integrated" analysis with extremely high sensitivity, which can be used for trace detection of early cancer. However, this sensor involves vacuum coating, precise self-assembly of multiple DNA strands, functionalization of nanoprobes, and other steps, which takes a long time and has high detection cost, limiting its large-scale application.

[0005] Chinese invention patent application CN201910642147.3 proposes an electrochemical aptamer sensor for detecting cancer marker CEA. It uses nickel-cobalt Prussian blue analog as electrode material, achieving ultra-high detection sensitivity of femtogram level, and good selectivity, stability and reproducibility. This method is a label-free detection, and the operation is relatively simple. However, the preparation of this sensor is still a multi-step laboratory process, which requires high control of conditions and may affect the consistency between batches. At the same time, the dependence on custom nucleic acid aptamer and special electrochemical instruments leads to high cost, making it difficult to realize large-scale instant detection application.

[0006] Therefore, there is an urgent need in the art for a new SERS detection strategy that can effectively suppress background signals, have signal amplification function, and achieve high specificity recognition of target substances, in order to meet the stringent requirements of detection sensitivity and accuracy for clinical early diagnosis. SUMMARY

[0007] The application aims to provide a lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform and a construction method and application thereof, so as to solve the problems of high background signal interference, great non-specific adsorption influence and low detection sensitivity in the prior art.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical scheme: In a first aspect, the application provides a lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform.

[0009] The lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform comprises: A first magnetic carrier complex comprising a first magnetic carrier on which a target protein aptamer is fixed, wherein the target protein aptamer is an aptamer specifically combined with a carcinoembryonic antigen protein CEA; A SERS probe comprising a gold nanoparticle, a surface of the gold nanoparticle being covalently connected with a Raman reporter molecule sequence and a capture DNA, wherein the SERS probe forms a DNA double-stranded structure by hybridization with the target protein aptamer on the first magnetic carrier complex through the capture DNA; A second magnetic carrier complex comprising a second magnetic carrier on which an enrichment DNA is fixed, wherein the enrichment DNA sequence is completely complementary to the capture DNA.

[0010] Preferably, the aptamer is CEA-Aptamer, and the sequence thereof is Biotin-AAAAAAAAAA AAT ACC AGCTTA TTC AAT TTA TCA AT.

[0011] Preferably, the capture DNA is AuNPs-cDNA, and the sequence thereof is SH-AAAAAAAAAAT TGA TAA ATT G.

[0012] Preferably, the Raman label molecule sequence is AuNPs-ROX, and the sequence thereof is ROX-TTT TTT CCT AGCGAC-SH.

[0013] Preferably, the enrichment DNA is MB-cDNA, and the sequence thereof is Biotin-CAATTTATCAATTTTTT.

[0014] Preferably, the CEA-Aptamer is modified by 5'-biotin, and is connected with an avidin-coated first magnetic carrier.

[0015] Preferably, the MB-cDNA is modified by biotin and connected to the second magnetic carrier coated with avidin.

[0016] Preferably, the capture DNA and Raman label molecule sequence on the SERS probe are both connected to the surface of gold nanoparticles through thiol modification.

[0017] In the second aspect, the application provides a method for constructing a magnetic bead enrichment SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion.

[0018] A method for constructing a magnetic bead enrichment SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion, comprising the following steps: (1) fixing the target protein aptamer on the surface of the streptavidin magnetic bead through biotin-avidin interaction to form a first magnetic carrier complex; (2) covalently modifying the Raman reporter molecule sequence and the capture DNA on the surface of the gold nanoparticles to form a SERS probe; (3) connecting the SERS probe to the first magnetic carrier complex through DNA hybridization to form a double-stranded structure; and (4) fixing the enrichment DNA on the surface of the streptavidin magnetic bead through biotin-avidin interaction to form a second magnetic carrier complex.

[0019] Preferably, in step (2), the preparation of the SERS probe comprises: (2.1) adding AuNPs-ROX and AuNPs-cDNA into a tris(2-carboxyethyl) phosphine hydrochloride (TCEP) solution, and rotating incubation at room temperature for 20-40 min to reduce the S-S bond; (2.2) taking the AuNPs solution and adding it to the reaction system obtained in step (2.1), shaking uniformly, and then quickly adding n-butanol and shaking until transparent to promote the interaction between the gold nanoparticles and the corresponding thiol-modified DNA and improve the adsorption rate; (2.3) taking 0.5×TBE buffer and adding it to the solution obtained in step (2.2), quickly stirring uniformly, and then centrifuging for 10 min, and then dispersing the precipitate in PBS buffer.

[0020] Preferably, in step (2.2), the preparation steps of the AuNPs solution are: directly adding a 1.0-1.2% mass concentration citric acid aqueous solution to a boiling HAuCl4 solution, continuing to stir for 5-15 min, and then cooling to room temperature.

[0021] In the third aspect, the application provides the application of the magnetic bead enrichment SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion.

[0022] The application discloses a lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform and an application thereof.

[0023] Preferably, the step of detecting the surface enhanced Raman spectrum of the lung cancer marker protein in the sample comprises the following steps: (a) incubating the sample to be detected with the double-stranded structure complex at room temperature; (b) collecting the supernatant containing the released SERS probe through magnetic separation; (c) making the supernatant react with a second magnetic carrier complex to capture the SERS probe; and (d) detecting the Raman signal intensity of the captured complex.

[0024] Compared with the prior art, the application has the following beneficial effects: The application provides a lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform, a construction method and an application thereof, and realizes super-sensitive and high-specificity quantitative detection of lung cancer marker proteins (such as CEA). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a flow chart of the application; Figure 2 is a schematic diagram of lung cancer marker protein detection based on signal conversion SERS probe; Figure 3 is a gold nanoparticle characterization result graph, wherein (a) is an ultraviolet absorption curve of gold nanosol; (b) is a TEM graph of gold nanoparticles; and (c) is a particle size distribution graph of gold nanoparticles; Figure 4 is an ultraviolet spectrum, wherein (a) is AuNPs-cDNA; (b) is AuNPs-ROX; (c) is AuNPs; and (d) is SERS-Probe; Figure 5 is a laser confocal Raman spectrum of a control experiment, wherein (a) is incubation without adding CEA; and (b) is incubation with the addition of CEA; Figure 6 is a Raman spectrum quantitative analysis result graph of CEA detection, wherein (a) is a SERS Raman spectrum graph under different concentration conditions; and (b) is a linear relationship graph of Raman intensity and different CEA concentrations. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below with reference to the drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0027] The overall flow chart of the application is shown in Figure 1 .

[0028] The application discloses a lung cancer marker protein-DNA signal conversion-based magnetic bead enrichment SERS probe sensing platform. The first magnetic carrier complex comprises a first magnetic carrier on which a target protein aptamer is fixed, and the target protein aptamer is an aptamer specifically combined with a carcinoembryonic antigen protein CEA. The SERS probe comprises gold nanoparticles, the surface of the gold nanoparticles is covalently connected with a Raman reporter molecule sequence and a capture DNA, and the SERS probe is hybridized with the target protein aptamer on the first magnetic carrier complex through the capture DNA to form a DNA double-stranded structure. The second magnetic carrier complex comprises a second magnetic carrier on which an enrichment DNA is fixed, and the enrichment DNA sequence is completely complementary to the capture DNA.

[0029] In some preferred embodiments of the application, the aptamer is CEA-Aptamer, and the sequence is Biotin-AAAAAAAAAA AAT ACC AGC TTA TTC AAT TTA TCA AT.

[0030] In some preferred embodiments of the application, the capture DNA is AuNPs-cDNA, and the sequence is SH-AAAAAAAAAATTG A TAA ATT G.

[0031] In some preferred embodiments of the application, the Raman label molecule sequence is AuNPs-ROX, and the sequence is ROX-TTTTTT CCT AGC GAC-SH.

[0032] In some preferred embodiments of the application, the enrichment DNA is MB-cDNA, and the sequence is Biotin-CAATTT ATCAAT TTT TT.

[0033] In some preferred embodiments of the application, the CEA-Aptamer is modified by 5'-biotin, and is connected with the first magnetic carrier coated with avidin.

[0034] In some preferred embodiments of the application, the MB-cDNA is modified by biotin, and is connected with the second magnetic carrier coated with avidin.

[0035] In some preferred embodiments of the application, the capture DNA and the Raman label molecule sequence on the SERS probe are both modified by thiol and connected with the surface of the gold nanoparticles.

[0036] The construction method of the SERS probe sensing platform based on the lung cancer marker protein-DNA signal conversion of the application comprises the following steps: (1) target protein aptamer is fixed on the surface of streptavidin magnetic beads through biotin-avidin interaction to form a first magnetic carrier complex; (2) a Raman reporter molecule sequence and a capture DNA are covalently modified on the surface of gold nanoparticles to form a SERS probe; (3) the SERS probe is connected to the first magnetic carrier complex through DNA hybridization to form a double-stranded structure; and (4) the enrichment DNA is fixed on the surface of streptavidin magnetic beads through biotin-avidin interaction to form a second magnetic carrier complex.

[0037] In some preferred embodiments of the application, in step (2), the preparation of the SERS probe comprises: (2.1) AuNPs-ROX and AuNPs-cDNA are added to a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution, and the S-S bond is reduced by rotating incubation at room temperature for 20-40 min; (2.2) AuNPs solution is added to the reaction system obtained in step (2.1), and after shaking uniformly, n-butanol is quickly added and shaken until transparent to promote the interaction of gold nanoparticles and the corresponding thiol-modified DNA and improve the adsorption rate; (2.3) 0.5×TBE buffer solution is added to the solution obtained in step (2.2), and after quick stirring, centrifugation is performed for 10 min, and the precipitate is dispersed with PBS buffer solution.

[0038] In some preferred embodiments of the application, in step (2.2), the preparation steps of the AuNPs solution are as follows: a 1.0-1.2% mass concentration citric acid aqueous solution is directly added to a boiling HAuCl4 solution, and after continuing to stir for 5-15 min, the solution is cooled to room temperature.

[0039] The SERS probe sensing platform based on the lung cancer marker protein-DNA signal conversion of the application can be applied to the preparation of a product for detecting the surface-enhanced Raman spectrum of lung cancer marker protein in a sample.

[0040] In some preferred embodiments of the application, the step of detecting the surface-enhanced Raman spectrum of lung cancer marker protein in a sample comprises: (a) incubating the sample to be detected with the double-stranded structure complex at room temperature; (b) collecting the supernatant containing the released SERS probe by magnetic separation; (c) allowing the supernatant to react with the second magnetic carrier complex to capture the SERS probe; and (d) detecting the Raman signal intensity of the captured complex.

[0041] Example 1: SERS probe detection of CEA protein based on signal conversion This example describes in detail the whole process of preparing the sensing platform and detecting carcinoembryonic antigen (CEA) by using the application, as follows:Figure 2 as shown.

[0042] Chemicals: Streptavidin magnetic beads (Beaver Streptavidin, 300 nm, 10 mg / mL); recombinant human carcinoembryonic antigen (CEA) protein; chloroauric acid tetrahydrate (HAuCl4·4H2O) and trisodium citrate were purchased from Bidepherm Company; tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from J&K Scientific Company. All reagents were of analytical purity.

[0043] Nucleic acid sequences: purchased from Sangon Biotech Company, see Table 1 for specific sequences.

[0044] Table 1 This example includes the following steps: 1. Preparation of gold nanoparticles (AuNPs) A 100 mL solution of chloroauric acid tetrahydrate with a concentration of 1 mM / L was added to a 250 mL three-necked flask, and heated to boiling in an oil bath. 2.6 mL of a 1% mass concentration of trisodium citrate solution was quickly added, and heating was continued while observing the color change. When the solution color changed from colorless to blue-black and finally stabilized to wine red, heating was stopped, and the solution was naturally cooled to room temperature. The prepared AuNPs were characterized by ultraviolet-visible spectrophotometry (a) and transmission electron microscopy (TEM) (b) to confirm that the characteristic absorption peak was near 520 nm, the particle size was about 15 nm, and the distribution was uniform (c). Figure 3 Figure 3 Figure 3

[0045] 2. Preparation of SERS probes 3 μL of AuNPs-ROX with a concentration of 100 μM and 3 μL of AuNPs-cDNA with a concentration of 10 μM were added to 14 μL of a TCEP solution with a concentration of 1 mM, and incubated at room temperature for 30 minutes to reduce the disulfide bond. Subsequently, the mixture was added to 80 μL of an AuNPs colloidal solution, shaken to mix, and then 900 μL of n-butanol was quickly added and shaken vigorously to promote the binding of DNA to the Au-S bond on the surface of AuNPs. After adding 20 μL of 0.5×TBE buffer (pH 8.0), centrifugation was performed at 9000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed three times with PBS buffer (pH 7.4), and finally the obtained SERS probe was resuspended in 40 μL of PBS buffer and stored at 4°C for standby. Ultraviolet-visible spectroscopy (a) and transmission electron microscopy (b) were used to characterize the prepared SERS probe, and the characteristic absorption peak was near 520 nm, the particle size was about 15 nm, and the distribution was uniform (c). Figure 4 ​​​) shows that the prepared SERS probe (curve d) has an absorption peak at 605 nm contributed by the ROX dye and an absorption peak at 260 nm contributed by the DNA, based on the 520 nm absorption peak of the AuNPs, proving that the SERS probe is successfully prepared.

[0046] 3. Preparation of the initial double-stranded DNA probe complex (1) Preparation of the first magnetic carrier modified with CEA-aptamer: Take 30 μL of streptavidin magnetic beads, wash them twice with Buffer I solution (Buffer I solution contains: 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20, pH 7.5). Dilute 5 μL of CEA-aptamer with a concentration of 100 μM to 150 μL with Buffer I, mix with the washed magnetic beads, and incubate at room temperature for 30 minutes. Remove the supernatant by magnetic separation, wash with Buffer I three times, and PBS twice, and finally resuspend in 200 μL of PBS to prepare the first magnetic carrier modified with CEA-aptamer.

[0047] (2) Construction of double-stranded probe: Take 2 μL of the above CEA-aptamer modified magnetic bead solution, discard the supernatant after magnetic separation. Add 8 μL of the SERS probe solution prepared in step 2 to the magnetic beads, use PBS as the hybridization buffer, and incubate at 45°C for 3 hours with gentle rotation. After cooling to room temperature, remove the unbound SERS probe by magnetic separation and wash with hybridization buffer three times to obtain the initial double-stranded DNA probe complex.

[0048] 4. Preparation of capture magnetic beads (second magnetic carrier) This step is similar to step 3 (1), replacing CEA-aptamer with 5 μL of MB-cDNA with a concentration of 100 μM, and the remaining operation steps are the same, to prepare the second magnetic carrier modified with MB-cDNA for subsequent probe capture.

[0049] 5. Detection of CEA (1) Probe release: In a centrifuge tube containing an equal amount of the initial double-stranded DNA probe complex, add PBS buffer containing different concentrations of CEA (e.g. 0, 1, 2, 5, 10 ng / mL). Incubate at room temperature for 2 hours.

[0050] (2) Probe enrichment and detection: After the incubation, separate the beads using a magnetic separator for 3 minutes and carefully aspirate all the supernatant. Transfer the supernatant to a pre-prepared centrifuge tube containing a second magnetic carrier modified with MB-cDNA and incubate for 3 hours. Then centrifuge at 1000 rpm, discard the supernatant, and wash the precipitate with a small amount of PBS. Drop the final precipitate (magnetic beads enriched with SERS probes) onto a clean gold sheet and dry at room temperature. Detection was performed using a laser confocal Raman spectrometer with an excitation wavelength of 633 nm and Raman spectra collected in the range of 600-2200 cm⁻¹.

[0051] Feasibility verification: Figure 5 As shown, the control group without CEA (curve a) detected a very weak Raman signal, indicating that in the absence of a target, the SERS probe was firmly locked to the first magnetic carrier and the system was in an "off" state. In contrast, the experimental group with CEA (curve b) detected a very strong ROX Raman characteristic peak, demonstrating that the addition of CEA successfully triggered the release of the SERS probe, achieving a signal transition from "off" to "on," validating the feasibility of this method.

[0052] Sensitivity and quantitative analysis: Figure 6 As shown in Figure a, as the CEA concentration increases (1 ng / mL to 10 ng / mL), the intensity of the ROX characteristic Raman peak at 1502 cm⁻¹ also increases systematically. The peak intensity at 1502 cm⁻¹ was linearly fitted with the CEA concentration, as shown in Figure 4. Figure 6 As shown in (b), a good linear relationship was obtained, with the linear regression equation being y = 1123.48 + 736.82x, and a high linear correlation coefficient (R²) of 0.9989. This demonstrates that the method of the present invention has excellent quantitative detection capability and high sensitivity within the concentration range of 1-10 ng / mL. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion, characterized by: include: A first magnetic carrier complex comprises a first magnetic carrier having a target protein aptamer immobilized thereon, wherein the target protein aptamer is an aptamer that specifically binds to the carcinoembryonic antigen protein CEA; SERS probes: These consist of gold nanoparticles with Raman reporter sequences and capture DNA covalently attached to their surfaces; The SERS probe hybridizes with the target protein aptamer on the first magnetic carrier complex by capturing DNA to form a DNA double-stranded structure; The second magnetic carrier complex comprises a second magnetic carrier on which the enriched DNA is immobilized, wherein the enriched DNA sequence is completely complementary to the captured DNA.

2. The magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion according to claim 1, characterized in that: The aptamer is CEA-Aptamer, and its sequence is Biotin-AAAAAAAAAA AAT ACC AGC TTA TTC AATTTA TCA AT; The captured DNA is AuNPs-cDNA, and its sequence is SH-AAAAAAAAAAT TGA TAA ATT G; The Raman tag molecular sequence is AuNPs-ROX, and its sequence is ROX-TTT TTT CCT AGC GAC-SH); The enriched DNA is MB-cDNA, and its sequence is Biotin-CAA TTT ATC AAT TTT TT.

3. The magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion according to claim 2, characterized in that: The CEA-Aptamer is modified with 5'-biotin and connected to a first magnetic carrier coated with avidin; the MB-cDNA is modified with biotin and connected to a second magnetic carrier coated with avidin.

4. The magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion according to claim 1 or 2, characterized in that: The capture DNA and Raman tag molecular sequences on the SERS probe are both connected to the surface of the gold nanoparticles through thiol modification.

5. A method for constructing a magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion according to claim 1, characterized in that: The following steps are involved: (1) The target protein aptamer is fixed to the surface of streptavidin magnetic beads through biotin-avidin interaction to form a first magnetic carrier complex; (2) The Raman reporter molecule sequence and capture DNA are covalently modified on the surface of gold nanoparticles to form a SERS probe; (3) The SERS probe is connected to the first magnetic carrier complex through DNA hybridization to form a double-stranded structure; (4) The enriched DNA is fixed to the surface of streptavidin magnetic beads through biotin-avidin interaction to form a second magnetic carrier complex.

6. The construction method according to claim 5, characterized in that: In step (2), the preparation of the SERS probe includes: (2.1) Add AuNPs-ROX and AuNPs-cDNA to tris(2-carboxyethyl)phosphine hydrochloride solution and incubate with rotation at room temperature for 20-40 min to reduce the SS bond; (2.2) Add the AuNPs solution to the reaction system obtained in step (2.1), shake evenly, and then quickly add n-butanol and shake until transparent to promote the interaction between the gold nanoparticles and the corresponding thiol-modified DNA, thereby improving the adsorption rate; (2.3) Add 0.5× TBE buffer to the solution obtained in step (2.2), stir quickly and evenly, then centrifuge for 10 min. Disperse the precipitate with PBS buffer.

7. The construction method according to claim 6, characterized in that: In step (2.2), the preparation steps of the AuNPs solution are as follows: directly add a 1.0-1.2% mass concentration of citric acid aqueous solution to the boiling HAuCl4 solution, continue stirring for 5-15 minutes, and then cool to room temperature.

8. Use of the magnetic bead-enriched SERS probe sensing platform based on lung cancer marker protein-DNA signal conversion according to claim 1 in preparing a surface-enhanced Raman spectroscopy product for detecting lung cancer marker proteins in a sample.

9. The use according to claim 8, characterized in that: The steps of detecting lung cancer marker proteins in a sample by surface-enhanced Raman spectroscopy include: (a) incubating the sample to be tested with the double-stranded structure complex at room temperature; (b) collecting the supernatant containing the released SERS probe by magnetic separation; (c) allowing the supernatant to react with a second magnetic carrier complex to capture the SERS probe; and (d) detecting the Raman signal intensity of the captured complex.

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