Sample separation chip, sample detection device and sample detection method

Abstract

The present invention provides a sample separation chip, a sample detection device, and a sample detection method. The sample separation chip includes a first substrate, a first electrode, a first dielectric layer, a second substrate, a second electrode, a second dielectric layer, and a channel layer. The first electrode is arranged on the first substrate. The first dielectric layer is arranged on the first electrode and includes a first opening. The first opening exposes a portion of the first electrode. The second electrode is arranged on the second substrate. The second dielectric layer is arranged on the second electrode and includes a second opening. The second opening exposes a portion of the second electrode. The area of ​​the first electrode exposed by the first opening is smaller than the area of ​​the second electrode exposed by the second opening. The channel layer is sandwiched between the first dielectric layer and the second dielectric layer and includes a through hole. The through hole is connected between the first opening and the second opening. The above-mentioned sample separation chip helps to shorten the sample detection process and detection time.

Description

Technical Field

[0001] The present invention relates to a chip, a detection device and a detection method, and in particular to a sample separation chip, a sample detection device and a sample detection method using surface-enhanced Raman spectroscopy. Background Art

[0002] Currently, pathogen analysis often delays optimal treatment due to complex testing processes and lengthy testing times. For example, bacterial isolation is traditionally performed using centrifugation, immunohistochemical targeting, and dialysis. However, these methods require lengthy wait times and complex techniques to achieve effective separation. Summary of the Invention

[0003] The present invention provides a sample separation chip, a sample detection device, and a sample detection method using surface-enhanced Raman spectroscopy, which are helpful in shortening the sample detection process and detection time.

[0004] The present invention provides a sample separation chip, comprising a first substrate, a first electrode, a first dielectric layer, a second substrate, a second electrode, a second dielectric layer, and a channel layer. The first electrode is disposed on the first substrate. The first dielectric layer is disposed on the first electrode and includes a first opening. The first opening exposes a portion of the first electrode. The second electrode is disposed on the second substrate. The second dielectric layer is disposed on the second electrode and includes a second opening. The second opening exposes a portion of the second electrode. The area of ​​the first electrode exposed by the first opening is smaller than the area of ​​the second electrode exposed by the second opening. The channel layer is sandwiched between the first dielectric layer and the second dielectric layer and includes a through hole. The through hole connects the first opening and the second opening.

[0005] According to an embodiment of the present invention, in the sample separation chip, the first opening, the second opening, and the through hole may be aligned with each other.

[0006] According to an embodiment of the present invention, in the sample separation chip, a top view area of ​​the through hole may be larger than a top view area of ​​the first opening.

[0007] According to an embodiment of the present invention, in the sample separation chip, a top view area of ​​the through hole may be greater than or equal to a top view area of ​​the second opening.

[0008] According to an embodiment of the present invention, in the sample separation chip, the number of the first opening may be one.

[0009] According to an embodiment of the present invention, in the sample separation chip, the number of the first openings may be plural.

[0010] According to an embodiment of the present invention, in the sample separation chip, the number of the second opening may be one.

[0011] According to an embodiment of the present invention, in the sample separation chip, the number of the second openings may be plural.

[0012] According to an embodiment of the present invention, in the sample separation chip, the top-view shapes of the first opening, the second opening, and the through-hole can be circular, polygonal, irregular, or a combination thereof.

[0013] According to an embodiment of the present invention, in the sample separation chip, the first dielectric layer may further include a third opening, wherein the third opening may expose another portion of the first electrode.

[0014] According to an embodiment of the present invention, in the sample separation chip, the second dielectric layer may further include a fourth opening, wherein the fourth opening may expose another portion of the second electrode.

[0015] According to an embodiment of the present invention, in the sample separation chip, the material of the first electrode and the material of the second electrode can be indium tin oxide (ITO), metal, conductive carbon material or a combination thereof.

[0016] According to an embodiment of the present invention, in the sample separation chip, the thickness of the channel layer may range from 20 μm to 100 μm.

[0017] According to an embodiment of the present invention, in the sample separation chip, the material of the channel layer may be a light-transmitting dielectric material.

[0018] The present invention provides a sample detection device comprising a Raman spectrometer, the sample separation chip, and an AC power supply. The separation chip is disposed in the Raman spectrometer. The AC power supply is electrically connected to a first electrode and a second electrode.

[0019] The present invention provides a sample detection method using surface-enhanced Raman spectroscopy, comprising the following steps: providing the above-mentioned sample separation chip; providing a sample liquid containing a biological sample to be tested into a channel formed by a first opening, a second opening, and a through hole; providing an alternating current to the first electrode and the second electrode, and separating and concentrating the biological sample to be tested in the sample liquid by electroosmotic flow (EOF) and dielectrophoresis force (DEP force); obtaining a surface-enhanced Raman spectrum of the separated and concentrated biological sample to be tested by a Raman spectrometer; and determining the type of the biological sample to be tested by the surface-enhanced Raman spectrum of the biological sample to be tested.

[0020] According to one embodiment of the present invention, in the above-mentioned sample detection method using surface-enhanced Raman spectroscopy, the Raman spectrum of the biological sample to be tested can be enhanced by adding metal particles to the sample liquid or making at least one of the first electrode and the second electrode have a rough metal surface, so as to obtain the surface-enhanced Raman spectrum of the biological sample to be tested.

[0021] According to one embodiment of the present invention, in the sample detection method using surface-enhanced Raman spectroscopy, the method for determining the type of a biological sample to be tested based on its surface-enhanced Raman spectrum may include the following steps: The surface-enhanced Raman spectrum of the biological sample to be tested is compared with a library of standard surface-enhanced Raman spectra to determine the type of the biological sample to be tested. The library of standard surface-enhanced Raman spectra may include multiple standard surface-enhanced Raman spectra corresponding to multiple standard biological samples.

[0022] According to one embodiment of the present invention, the sample detection method using surface-enhanced Raman spectroscopy may further include the following steps: After determining the type of the biological sample to be tested, performing an antimicrobial susceptibility test (AST) on the biological sample to be tested. The method for antimicrobial susceptibility testing may include the following steps: adding an antibiotic to the sample solution. After adding the antibiotic to the sample solution, measuring the surface-enhanced Raman spectrum of the biological sample to be tested.

[0023] According to an embodiment of the present invention, in the sample detection method using surface-enhanced Raman spectroscopy, the frequency of the alternating current may range from 500 Hz to 14 MHz.

[0024] Based on the above, in the sample separation chip, sample detection device, and sample detection method using surface-enhanced Raman spectroscopy proposed in the present invention, the area of ​​the first electrode exposed by the first opening is smaller than the area of ​​the second electrode exposed by the second opening. Thus, after supplying alternating current to the first and second electrodes, the change in the electric field gradient at the first electrode exposed by the first opening is greater than the change in the electric field gradient at the second electrode exposed by the second opening. Therefore, the biological sample to be tested located in the channel of the sample separation chip can be rapidly separated and concentrated through electroosmotic flow and dielectrophoretic force, thereby helping to shorten the sample detection process and detection time.

[0025] In order to make the above features and advantages of the present invention more clearly understood, embodiments are given below with reference to the accompanying drawings for detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A is an exploded view of a sample separation chip according to an embodiment of the present invention;

[0027] Figure 1B is a diagram showing an assembly of a sample separation chip according to an embodiment of the present invention;

[0028] Figure 1C for Figure 1A A top view of the components of the sample separation chip;

[0029] Figure 1D To follow Figure 1B The cross-sectional view along the section line I-I';

[0030] Figure 2 is a schematic diagram of a sample detection device according to an embodiment of the present invention;

[0031] Figure 3 A flow chart of sample detection using surface-enhanced Raman spectroscopy according to an embodiment of the present invention;

[0032] Figure 4 A schematic diagram of separating and concentrating a biological sample to be tested in a sample liquid by electroosmotic flow and dielectrophoretic force according to an embodiment of the present invention;

[0033] Figure 5 is a graph showing the relationship between the alternating current frequency (AC (alternating current) frequency) and the relative dielectrophoretic force constant in an experimental example of the present invention;

[0034] Figure 6 This is a graph showing the relationship between the AC frequency and the bacterial intensity in a concentrated area in an experimental example of the present invention;

[0035] Figure 7is a graph showing the relationship between the alternating current application time and the bacterial intensity in the concentrated area in an experimental example of the present invention;

[0036] Figure 8 This is a surface-enhanced Raman spectrum diagram of an experimental example of the present invention.

[0037] Description of Figure Numbers:

[0038] 10: Sample detection device

[0039] 100: Sample separation chip

[0040] 102,108:Substrate

[0041] 104,110: Electrode

[0042] 106,112: Dielectric layer

[0043] 114: Channel layer

[0044] 200:Raman spectrometer

[0045] 300: AC power supply unit

[0046] ACEOF: Alternating Current Electroosmotic Flow

[0047] C: Channel

[0048] nDEP: Negative electrophoretic force

[0049] OP1~OP4: Open

[0050] pDEP: Positive Electrophoretic Force

[0051] H:Through hole

[0052] S1: Biological sample to be tested

[0053] S2: non-test samples

[0054] S100, S102, S104, S106, S108, S110: Steps

[0055] SS: sample solution DETAILED DESCRIPTION

[0056] Figure 1A FIG. 4 is an exploded view of a sample separation chip according to an embodiment of the present invention. Figure 1B FIG. 4 is a diagram showing an assembly of a sample separation chip according to an embodiment of the present invention. Figure 1C for Figure 1A Top view of the components of the sample separation chip. Figure 1D To follow Figure 1B Sectional view along section line I-I'.

[0057] Please refer to Figures 1A to 1D The sample separation chip 100 includes a substrate 102, an electrode 104, a dielectric layer 106, a substrate 108, an electrode 110, a dielectric layer 112, and a channel layer 114. In some embodiments, the sample separation chip 100 is, for example, a biochip for separating biological samples. The materials of the substrate 102 and the substrate 108 can each be a dielectric material such as glass.

[0058] The electrode 104 is disposed on the substrate 102. The material of the electrode 104 can be indium tin oxide, metal, conductive carbon material, or a combination thereof. The electrode 104 can be formed on the substrate 102 by physical vapor deposition or chemical vapor deposition.

[0059] The dielectric layer 106 is disposed on the electrode 104 and includes an opening OP1. The opening OP1 exposes a portion of the electrode 104. The number of openings OP1 may be one or more. In this embodiment, the number of openings OP1 is shown as a plurality and is not limited to the number shown in the figure. The shapes and areas of the plurality of openings OP1 may be the same or different. The top view shape of the opening OP1 may be circular, polygonal, irregular, or a combination thereof. In this embodiment, the top view shape of the opening OP1 is shown as a circle, but the present invention is not limited thereto. In addition, the dielectric layer 106 may further include an opening OP2. The opening OP2 may expose another portion of the electrode 104. The electrode 104 exposed by the opening OP2 may be used to electrically connect to a power source (e.g., an AC power source). The top view shape of the opening OP2 may be circular, polygonal, or irregular. In this embodiment, the top view shape of the opening OP2 is shown as a rectangle, but the present invention is not limited thereto. The thickness of the dielectric layer 106 may range from 200 nanometers (nm) or more. In addition, the dielectric layer 106 having the opening OP1 and the opening OP2 can be formed on the electrode 104 through a deposition process, a photolithography process, and an etching process.

[0060] The electrode 110 is disposed on the substrate 108. The material of the electrode 110 can be indium tin oxide, metal, conductive carbon material, or a combination thereof. The electrode 110 can be formed on the substrate 108 by physical vapor deposition or chemical vapor deposition.

[0061] The dielectric layer 112 is disposed on the electrode 110 and includes an opening OP3. The opening OP3 exposes a portion of the electrode 110. The number of openings OP3 may be one or more. In this embodiment, the number of openings OP3 is shown as one, but the present invention is not limited thereto. In other embodiments, the number of openings OP3 may be multiple, and the shapes and areas of the multiple openings OP3 may be the same or different. The top-view shape of the opening OP3 may be circular, polygonal, irregular, or a combination thereof. In this embodiment, the top-view shape of the opening OP3 is shown as a circular shape, but the present invention is not limited thereto. In addition, the dielectric layer 112 may further include an opening OP4. The opening OP4 may expose another portion of the electrode 110. The electrode 110 exposed by the opening OP4 can be used to electrically connect to a power source (e.g., an AC power source). The top-view shape of the opening OP4 may be circular, polygonal, or irregular. In this embodiment, the top-view shape of the opening OP4 is shown as a rectangular shape, but the present invention is not limited thereto. The thickness of the dielectric layer 112 may range from 200 nm or more. In addition, the dielectric layer 112 having the opening OP3 and the opening OP4 may be formed on the electrode 110 through a deposition process, a photolithography process, and an etching process.

[0062] Furthermore, the area of ​​the electrode 104 exposed by the opening OP1 is smaller than the area of ​​the electrode 110 exposed by the opening OP3. Therefore, after applying alternating current to the electrodes 104 and 110, the electric field gradient change at the electrode 104 exposed by the opening OP1 may be greater than the electric field gradient change at the electrode 110 exposed by the opening OP3. In some embodiments, when there are multiple openings OP1, the "area of ​​the electrode 104 exposed by the opening OP1" refers to the "total area of ​​the electrode 104 exposed by all openings OP1." In some embodiments, when there are multiple openings OP3, the "area of ​​the electrode 110 exposed by the opening OP3" refers to the "total area of ​​the electrode 110 exposed by all openings OP3."

[0063] The channel layer 114 is sandwiched between the dielectric layer 106 and the dielectric layer 112, and includes a through hole H. The through hole H is connected between the opening OP1 and the opening OP3. The top view shape of the through hole H can be circular, polygonal, irregular, or a combination thereof. In the present embodiment, the top view shape of the through hole H is a circle, but the present invention is not limited to this. The thickness of the channel layer 114 can range from 20 μm to 100 μm. The material of the channel layer 114 can be a light-transmitting dielectric material, such as polydimethylsiloxane (PDMS) or other non-conductive materials. In addition, a mold can be made using a photolithography process and an etching process, and then a light-transmitting dielectric material can be used for remolding to form the channel layer 114.

[0064] In addition, the opening OP1, the opening OP3 and the through hole H can be aligned with each other. The top viewing area of ​​the through hole H can be larger than the top viewing area of ​​the opening OP1. The top viewing area of ​​the through hole H can be greater than or equal to the top viewing area of ​​the opening OP3. In this embodiment, the top viewing area of ​​the through hole H is larger than the top viewing area of ​​the opening OP3, but the present invention is not limited to this. In addition, please refer to Figure 1D After the substrate 102 provided with the electrodes 104 and the dielectric layer 106, the substrate 108 provided with the electrodes 110 and the dielectric layer 112, and the channel layer 114 are combined, the openings OP1, OP3, and the through-holes H can form a channel C. In some embodiments, the channel C can serve as a microchannel of the biochip. Alternatively, the substrate 102 provided with the electrodes 104 and the dielectric layer 106, the substrate 108 provided with the electrodes 110 and the dielectric layer 112, and the channel layer 114 can be combined by clamping or bonding to form the sample separation chip 100.

[0065] Figure 2 FIG. 1 is a schematic diagram of a sample detection device according to an embodiment of the present invention.

[0066] Please refer to Figure 2 The sample detection device 10 may include a Raman spectrometer 200, the sample separation chip 100, and an AC power supply device 300. When performing sample detection, the separation chip 100 may be placed in the Raman spectrometer 200. In addition, when performing sample detection, the AC power supply device 300 is electrically connected to the electrodes 104 and 110 to provide AC power to the electrodes 104 and 110.

[0067] Figure 3 FIG. 4 is a flow chart of sample detection using surface-enhanced Raman spectroscopy according to an embodiment of the present invention. Figure 4 FIG. 1 is a schematic diagram of separating and concentrating a biological sample to be tested in a sample liquid by electroosmotic flow and dielectrophoretic force according to an embodiment of the present invention.

[0068] Please refer to Figures 1A to 1D and Figures 2 to 4 , step S100 is performed to provide a sample separation chip 100. For details of the sample separation chip 100, please refer to the description of the above embodiment, which will not be described again here.

[0069] Next, step S102 is performed to mix the sample solution SS ( Figure 4 ) is provided to the channel C formed by the opening OP1, the opening OP3 and the through hole H. Figure 4As shown, the sample liquid SS may contain not only the biological sample S1 to be tested but also a non-test sample S2. For example, the sample liquid SS may be blood, the biological sample S1 to be tested may be bacteria (e.g., E. coli), and the non-test sample S2 may include white blood cells (WBC) and red blood cells (RBC), but the present invention is not limited thereto.

[0070] Then, step S104 is performed to provide alternating current to the electrodes 104 and 110, and the biological sample S1 to be tested in the sample liquid SS is separated and concentrated by electroosmotic flow and dielectrophoretic force. Figure 2 The AC power supply device 300 in the embodiment provides AC power to the electrodes 104 and 110. The frequency of the AC power may range from 500 Hz to 14 MHz. The voltage of the AC power may range from 1 volt (V) to 100 volts, which is limited by the resistance of the dielectric material used. In some embodiments, the voltage of the AC power applied to the electrodes 104 and 110 may be the same, and the frequency of the AC power applied to the electrodes 104 and 110 may be the same.

[0071] Since the area of ​​the electrode 104 exposed by the opening OP1 is smaller than the area of ​​the electrode 110 exposed by the opening OP3, after the alternating current is supplied to the electrodes 104 and 110, the electric field gradient change intensity at the electrode 104 exposed by the opening OP1 will be greater than the electric field gradient change intensity at the electrode 110 exposed by the opening OP3. The movement of a substance under the action of an inhomogeneous electric field will vary depending on the dielectric constant, size, and dielectric constant of the substance. Therefore, the biological sample S1 to be tested located in the channel C of the sample separation chip 100 can be quickly separated and concentrated by electroosmotic flow and dielectrophoretic force. For example, Figure 4 As shown, when the biological sample S1 to be tested is mainly affected by the alternating current electroosmotic flow ACEOF and the positive electrophoretic force pDEP, the biological sample S1 to be tested will be concentrated on the electrode 104 exposed by the opening OP1 due to the adsorption force. Figure 4 As shown, when the non-test sample S2 is mainly affected by the alternating electroosmotic flow ACEOF and the negative electrophoretic force nDEP, the non-test sample S2 will be moved away from the electrode 104 exposed by the opening OP1 due to the repulsive force. Therefore, the biological sample S1 in the sample liquid SS can be quickly separated and concentrated.

[0072] Figure 5 FIG. 4 is a graph showing the relationship between the alternating current frequency and the relative dielectrophoretic force constant in an experimental example of the present invention. Figure 6 FIG. 4 is a graph showing the relationship between the AC frequency and the bacterial intensity in a concentrated area in an experimental example of the present invention. Figure 7FIG. 4 is a graph showing the relationship between the alternating current application time and the bacterial intensity in the concentrated area in an experimental example of the present invention.

[0073] In an experimental example, the sample liquid SS may be blood, the biological sample S1 to be tested may be Escherichia coli (E.Coli), and the non-test sample S2 may be white blood cells and red blood cells. Figure 5 As shown in FIG, when the AC voltage used in the sample detection method is 5V and the AC frequency is 1kHz to 41kHz, E. coli is subjected to positive dielectrophoretic force, while white blood cells and red blood cells are subjected to negative dielectrophoretic force. Therefore, an AC frequency within the above AC frequency range can be selected to separate and concentrate E. coli. Figure 6 As shown in FIG, when the AC voltage used in the sample detection method is 5V and the AC frequency is 1kHz to 41kHz, a better separation and concentration effect of Escherichia coli can be achieved. Figure 7 As shown, when the AC voltage used in the sample detection method is 5V and the AC frequency is 5kHz, E. coli can be effectively separated and concentrated within a very short time (eg, within 10 seconds).

[0074] Please continue to refer to Figures 1A to 1D 、 Figure 2 and Figure 3 , proceed to step S106, and obtain the surface-enhanced Raman spectrum of the separated and concentrated biological sample S1 to be tested by the Raman spectrometer 200. For example, the sample separation chip 100 in the power-on state can be set in the surface-enhanced Raman spectrometer 200 to obtain the surface-enhanced Raman spectrum of the biological sample S1 to be tested. In this embodiment, "surface-enhanced Raman spectrum" refers to the spectrum obtained by surface-enhanced Raman scattering (SERS). In some embodiments, the Raman spectrum of the biological sample S1 to be tested can be enhanced by adding metal particles to the sample liquid SS or making at least one of the electrode 104 and the electrode 110 have a rough metal surface to obtain the surface-enhanced Raman spectrum of the biological sample S1 to be tested. The metal particles can be nanometer-scale particles. The material of the metal particles can be silver, gold, platinum, nickel, copper or a combination thereof.

[0075] Next, step S108 is performed to determine the category of the biological sample S1 to be tested by the surface-enhanced Raman spectrum of the biological sample S1 to be tested. For example, the method for determining the category of the biological sample S1 to be tested by the surface-enhanced Raman spectrum of the biological sample S1 to be tested may include the following steps. The surface-enhanced Raman spectrum of the biological sample S1 to be tested is compared with a standard surface-enhanced Raman spectrum database to determine the category of the biological sample S1 to be tested. The standard surface-enhanced Raman spectrum database may include multiple standard surface-enhanced Raman spectra corresponding to multiple standard biological samples. In some embodiments, the standard surface-enhanced Raman spectrum database may be stored in the memory of a computer system, and the surface-enhanced Raman spectrum of the biological sample S1 to be tested may be compared with the standard surface-enhanced Raman spectrum database through the computer system to determine the category of the biological sample S1 to be tested.

[0076] Figure 8 This is a surface-enhanced Raman spectrum diagram of an experimental example of the present invention.

[0077] like Figure 8 As shown in FIG, the surface enhanced Raman spectrum of the biological sample S1 (E. coli) separated and concentrated by the above sample detection method has a high similarity with the standard surface enhanced Raman spectrum of pure E. coli, so the type of the biological sample S1 can be determined to be E. coli. Figure 8 As shown in FIG, the surface enhanced Raman spectrum of the blood containing E. coli has a low similarity to the standard surface enhanced Raman spectrum of pure E. coli. Therefore, it is difficult to directly determine that the biological sample S1 to be tested is E. coli based on the surface enhanced Raman spectrum of the blood containing E. coli. Figure 8 As shown, the SERS spectrum of blood has a low similarity to the standard SERS spectrum of pure E. coli.

[0078] Please continue to refer to Figures 1A to 1D 、 Figure 2 and Figure 3, proceed to step S110. After determining the category of the biological sample S1 to be tested, an antimicrobial susceptibility test (AST) is performed on the biological sample S1 to be tested (e.g., pathogens such as bacteria). The method for antimicrobial susceptibility testing may include the following steps. First, an antibiotic is added to the sample liquid. Then, after the antibiotic is added to the sample liquid, the surface-enhanced Raman spectrum of the biological sample S1 to be tested is measured. For example, after the antibiotic is added to the sample liquid, the sample separation chip 100 in the energized state can be set in the surface-enhanced Raman spectrometer 200 to measure the surface-enhanced Raman spectrum of the biological sample S1 to be tested. After the antibiotic is added to the sample liquid, if the surface-enhanced Raman spectrum signal of the biological sample S1 to be tested disappears or decreases to a certain extent (e.g., decreases by more than 50%), it can be determined that the antibiotic is effective. Conversely, after the antibiotic is added to the sample liquid, if a strong surface-enhanced Raman spectrum signal of the biological sample S1 to be tested is still obtained, it can be determined that the antibiotic is ineffective.

[0079] Based on the above embodiments, it can be seen that in the sample separation chip 100, sample detection device 10, and sample detection method using surface-enhanced Raman spectroscopy, the area of ​​electrode 104 exposed by opening OP1 is smaller than the area of ​​electrode 110 exposed by opening OP3. As a result, after applying alternating current to electrodes 104 and 110, the electric field gradient change at electrode 104 exposed by opening OP1 is greater than the electric field gradient change at electrode 110 exposed by opening OP3. Therefore, the biological sample S1 to be tested, located in channel C of the sample separation chip 100, can be rapidly separated and concentrated by electroosmotic flow and dielectrophoretic force, thereby helping to shorten the sample detection process and detection time.

[0080] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Any person skilled in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A chip for sample separation, characterized in that: include: a first substrate; a first electrode, disposed on the first substrate; a first dielectric layer disposed on the first electrode and comprising a first opening, wherein the first opening exposes a portion of the first electrode; a second substrate; a second electrode, disposed on the second substrate; a second dielectric layer disposed on the second electrode and comprising a second opening, wherein the second opening exposes a portion of the second electrode, and an area of ​​the first electrode exposed by the first opening is smaller than an area of ​​the second electrode exposed by the second opening; as well as a channel layer, sandwiched between the first dielectric layer and the second dielectric layer, and comprising a through hole, wherein the through hole communicates between the first opening and the second opening; The first opening, the second opening and the through hole are aligned with each other, The top view area of ​​the through hole is greater than or equal to the top view area of ​​the second opening. A top-view area of ​​the through hole is larger than a top-view area of ​​the first opening.

2. The sample separation chip according to claim 1, characterized in that The number of the first opening is one.

3. The sample separation chip according to claim 1, characterized in that There are multiple first openings.

4. The sample separation chip according to claim 1, wherein The number of the second opening is one.

5. The sample separation chip according to claim 1, wherein There are multiple second openings.

6. The sample separation chip according to claim 1, wherein The top-view shapes of the first opening, the second opening, and the through hole include circular, polygonal, irregular, or a combination thereof.

7. The sample separation chip according to claim 1, characterized in that The first dielectric layer further includes a third opening, wherein the third opening exposes another portion of the first electrode.

8. The sample separation chip according to claim 1, characterized in that The second dielectric layer further includes a fourth opening, wherein the fourth opening exposes another portion of the second electrode.

9. The sample separation chip according to claim 1, wherein The material of the first electrode and the material of the second electrode respectively include indium tin oxide, metal, conductive carbon material or a combination thereof.

10. The sample separation chip according to claim 1, characterized in that The thickness of the channel layer ranges from 20 microns to 100 microns.

11. The sample separation chip according to claim 1, characterized in that The material of the channel layer includes a light-transmitting dielectric material.

12. A sample detection device, characterized in that: include: Raman spectrometer; The sample separation chip according to claim 1, arranged in the Raman spectrometer; as well as The AC power supply device is electrically connected to the first electrode and the second electrode.

13. A sample detection method using surface enhanced Raman spectroscopy, characterized in that: include: Providing the sample separation chip according to claim 1; Providing a sample liquid containing a biological sample to be tested into the channel formed by the first opening, the second opening, and the through hole; providing alternating current to the first electrode and the second electrode, thereby separating and concentrating the biological sample to be tested in the sample liquid through electroosmotic flow and dielectrophoretic force; Obtaining a surface-enhanced Raman spectrum of the separated and concentrated biological sample to be tested by a Raman spectrometer; as well as The type of the biological sample to be tested is determined by the surface enhanced Raman spectrum of the biological sample to be tested.

14. The sample detection method using surface enhanced Raman spectroscopy according to claim 13, characterized in that: The Raman spectrum of the biological sample to be tested is enhanced by adding metal particles to the sample liquid or making at least one of the first electrode and the second electrode have a rough metal surface, so as to obtain the surface-enhanced Raman spectrum of the biological sample to be tested.

15. The sample detection method using surface enhanced Raman spectroscopy according to claim 13, characterized in that: The method for determining the type of the biological sample to be tested by the surface-enhanced Raman spectroscopy of the biological sample to be tested includes: The surface-enhanced Raman spectrum of the biological sample to be tested is compared with a standard surface-enhanced Raman spectrum database to determine the type of the biological sample to be tested, wherein the standard surface-enhanced Raman spectrum database includes a plurality of standard surface-enhanced Raman spectra corresponding to a plurality of standard biological samples.

16. The sample detection method using surface enhanced Raman spectroscopy according to claim 13, characterized in that: The method further includes performing an antimicrobial susceptibility test on the biological sample to be tested after determining the type of the biological sample to be tested, wherein the method of the antimicrobial susceptibility test comprises: adding antibiotics to the sample solution; and After the antibiotic is added to the sample solution, the surface enhanced Raman spectrum of the biological sample to be tested is measured.

17. The sample detection method using surface enhanced Raman spectroscopy according to claim 13, characterized in that: The frequency of the alternating current is in the range of 500 Hz to 14 MHz.

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