FETAL CELL CAPTURE MODULE AND MICROFLUIDIC CHIP FOR FETAL CELL CAPTURE AND METHODS FOR USING THE SAME
20230092810 · 2023-03-23
Inventors
- Chaoyong YANG (Xiamen, CN)
- Huimin ZHANG (Xiamen, CN)
- Yuanyuan YANG (Xiamen, CN)
- Yilong LIU (Xiamen, CN)
- Zhi ZHU (Xiamen, CN)
Cpc classification
B01L2200/0652
PERFORMING OPERATIONS; TRANSPORTING
C12N2509/00
CHEMISTRY; METALLURGY
B01L3/502761
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
The present invention relates to a fetal cell capture module, a microfluidic chip for fetal cell capture, and methods for using the same. The fetal cell capture module comprises a cell capture carrier and recognition molecule(s) for specific capture the cell(s). The recognition molecule is attached to the surface of the carrier via an organic conjugate L comprising disulfide bonds. The surface of the chip is modified with recognition molecules that specifically capture fetal cells via organic conjugates comprising disulfide bonds. The recognition molecule, after capturing the cell, achieves the release of the cell by chemically cleaving the disulfide bonds in the organic coupling conjugate. The present invention enables the capture of fetal cell(s) from whole blood without pre-treatment with a high capture rate, low cell loss, simple and accurate cell release operation, and the efficient and noninvasive release of fetal cells and whole genome analysis.
Claims
1-14. (canceled)
15. A fetal cell capture module, comprising a cell capture carrier and one or more recognition molecules for specific capture of cell(s), with the recognition molecule being attached to the surface of the cell capture carrier via an organic conjugate L comprising one or more disulfide bonds, wherein, the organic conjugate L has the formula:
-A-X—, wherein, A is selected from ##STR00014## with A being covalently linked to the capture carrier via the non-sulfur bond end, wherein n=1-10, f=1-10; preferably n=3-8 and f=2-8; X is selected from ##STR00015## preferably X is selected from ##STR00016## wherein the sulfur end of X is covalently attached to A to form a disulfide bond, with the other end of X being directly or indirectly connected to the recognition molecule, m=0-115, u=1-10; preferably, m=20-50, u=2-8.
16. The capture module according to claim 15, wherein, the organic conjugate L is selected from one or more of the following structures: ##STR00017## wherein, m=0-115; preferably, m=20-50.
17. The capture module according to claim 15, wherein the recognition molecule comprises one or more of a nucleic acid aptamer, a polypeptide, or an antibody; preferably, the recognition molecule is one or both of an anti-EpCAM antibody or an anti-CD71 antibody.
18. The capture module according to claim 15, wherein the fetal cell is a nucleated erythrocyte or a trophoblast cell.
19. The capture module according to claim 15, wherein the cell capture carrier includes a magnetic bead or a microfluidic chip.
20. A method of using the capture module according to claim 15, comprising bringing the capture module in contact with a liquid comprising fetal cell(s) to enable capture of the fetal cell(s); preferably, the liquid comprising fetal cell(s) comprises peripheral blood, cervical swab dispersion or suspension of a pregnant mammal or pregnant woman, or non-pregnant peripheral blood, buffer or culture solution comprising fetal cell(s); preferably, the liquid is directly contacted with the capture module without pre-isolation treatment; preferably, the capture module that has captured the fetal cell(s) is contacted with a chemical cleaving agent to break the disulfide bond of the organic coupling agent L to achieve the release of the fetal cell(s); preferably, the chemical cleaving agent is one or more of dithiothreitol, tris(2-carboxyethyl)phosphine, or glutathione.
21. A microfluidic chip for fetal cell capture, wherein the surface of the chip is modified via organic conjugate(s) L comprising one or more disulfide bonds by one or more recognition molecules that specifically capture fetal cell(s), wherein, the release of the cell being achieved by chemically cleaving the one or more disulfide bonds in the organic conjugate L after the cell being captured by the recognition molecule; preferably, the microfluidic chip is provided with one or more inlets, outlets and fluidic microchannels for fluid passage; preferably, the fluidic microchannel is further provided with a microarray which comprising a plurality of microcolumns arranged in one or more rows; preferably, the cross-sectional shape of the microcolumns is triangular; wherein, the organic conjugate L has the formula:
-A-X—, wherein, A is selected from ##STR00018## with A being covalently linked to the chip via the non-sulfur bond end wherein n=1-10, f=1-10; preferably, n=3-8 and f=2-8; X is selected from ##STR00019## preferably X is selected from ##STR00020## wherein the sulfur end of X is covalently attached to A to form a disulfide bond, with the other end of X being directly or indirectly connected to the recognition molecule, m=0-115, u=1-10; preferably, m=20-50, u=2-8.
22. The microfluidic chip according to claim 21, wherein, the organic conjugate L is selected from one or more of the following structures: ##STR00021## wherein, m=0-115; preferably, m=20-50.
23. The microfluidic chip according to claim 21, wherein the chemical cleaving is achieved by using one or more of dithiothreitol, tris(2-carboxyethyl)phosphine and glutathione.
24. The microfluidic chip according to claim 21, wherein the recognition molecule comprises one or more of a nucleic acid aptamer, a polypeptide, or an antibody; preferably, the recognition molecule is one or both of an anti-EpCAM antibody or an anti-CD71 antibody.
25. The microfluidic chip according to claim 21, wherein the fetal cell is a nucleated erythrocyte or a trophoblast cell.
26. A method of using the microfluidic chip according to any one of claim 21, comprising: (1) obtaining a liquid comprising fetal cell(s); (2) introducing the liquid obtained in step (1) into the microfluidic chip such that the fetal cell(s) in the liquid being brought into contacted with the one or more specific recognition molecules to realize the capture of the fetal cell(s); preferably, the method further comprising: (3) introducing a chemical cleavage agent to the microfluidic chip to break the one or more disulfide bonds in the organic conjugate(s) and release the captured fetal cell(s); preferably, the liquid comprising fetal cell(s) comprises peripheral blood, cervical swab dispersion or suspension of a pregnant mammal or pregnant woman, or non-pregnant peripheral blood, non-pregnant cervical swab dispersion or suspension, buffer or culture solution comprising fetal cell(s); preferably, introducing the liquid directly into the microfluidic chip without pre-isolation treatment; preferably, introducing the liquid through the microfluidic chip at a flow rate of 0.1-10 mL/h, preferably 0.1 to 1 mL/h.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
EMBODIMENTS
Example 1 Experimental Example of Magnetic Beads+Antibodies Modified by Disulfide Bonds
[0105] 1.1 Experimental Method
[0106] The magnetic beads were Dynabeads™ MyOne™ Carboxylic Acid obtained from Thermo Fisher with item number of 65012, and the method of using the magnetic beads is described in the instructions for use, which is shown as follows: The beads were removed, shaken and mixed, 20 L of the beads were washed three times with 15 mM MES buffer pH 6.0, a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added (see instruction for concentration), allowed to react for 30 min and magnetically separated, then washed 3 times with 15 mM MES buffer pH 6.0. 3-sulfhydryl-2-propanamine was added, allowed to react for 30 min, magnetically separated and washed 3 times with 15 mM MES buffer pH 6.0. A solution of 0.01% (mass fraction) of m-pyridyl disulfide polyethylene glycol succinimidyl valerate (with the polyethylene glycol molecular weight of 2000) was added, allowed to react for 30 min, magnetically separated, and washed e times with 15 mM MES buffer pH 6.0. 20 μg/mL of recognition antibodies were added, incubated for 1 h at room temperature, washed 3 times with 1×PBS+0.01% BSA, and immunomagnetic beads (resuspended in 20 μL of buffer) were obtained. 5 μL of immunomagnetic beads were mixed with 2 mL of peripheral blood of pregnant women, incubated for 45 min at room temperature, and magnetically separated. The cells obtained by magnetic separation were stained with antibodies and analyzed by fluorescence microscopy imaging. In which, the blood processing method is referred to step 3.1 in Example 3 below. After achieving cell capture, a solution of 100 μL of 50 mM DTT was introduced, allowed to react at 37° C. for 10 minutes, magnetically separated. The number of cells in the supernatant was counted, and cell activity was analyzed, referring to Example 4 4.1 for the specific steps, and the results are shown in Table 1.
2) Comparative Example 1
[0107] Experiments were performed with reference to the same methods as described above, with the difference that after cell capture, 100 μl of commercial trypsin (Thermo Fisher, Trypsin-EDTA (0.25%), phenol red, item number 25200056) was taken for cell release. The analysis of the viability of the released cells was performed referring to the method of Example 4 4.1.
[0108] 1.2 Experimental Results
TABLE-US-00001 TABLE 1 Comparison of disulfide bond chemical cleavage release and trypsin release methods with magnetic beads as cell capture carriers Release Recovery Purity prior to Purity after Example Efficiency Efficiency Release Release Cell Viability Example 1.1 85.30% ± 6.03 64.50% ± 6.18 2.22% ± 1.12 56.47% ± 9.36 85.60% ± 2.90 Comparative 69.75% ± 0.16 40.56% ± 5.90 1.23% ± 0.04 15.23% ± 1.05 53.6% ± 2.10 Example 1
Example 2 Preparation of Microfluidic Chip for Fetal Cell Capture
[0109] The microfluidic chip was fabricated with reference to the structure shown in
[0110] The chip is set with two inlets: inlet (1) and inlet (2), and one outlet: outlet (3), and a fluidic microchannel set with microarrays between the inlet and outlet, the structure of which is shown in
[0111] As a specific example (see
[0112] The coupling of recognition molecules can be performed by two methods, respectively, as follows:
[0113] Recognition molecule coupling method I: directly coupling the recognition molecule to the microchannel, as follows: the fluid microchannel of this specific example was irradiated by a plasma cleaner and immediately adhered to the carrier slide, and 20 μL ethanol solution of (3-mercaptopropyl)trimethoxysilane (MPTS) with a volume ratio of 4% was introduced every 5 minutes for 1 h. The operation can be performed by manual syringe injection or by automatic injection using an automatic microsampler, preferably Harvard Apparatus Pump 11 Pico Plus Elite syringe pump, US. Immediately afterwards, the channels were washed 3 times with ethanol solution, 100 μl each time, and placed in an oven for one hour at a heating temperature of 100° C. to obtain the highest efficiency of surface sulfhydrylation. The chip was taken out, cooled to room temperature and 0.01% (mass fraction) of o-pyridyl disulfide polyethylene glycol succinimidyl valerate (with the polyethylene glycol molecular weight of 2000) was introduced.
[0114] After 30 minutes of reaction, the microchannels were rinsed with deionized water and 3 times with PBS buffer, 100 μl each time, and then 20 μg/ml of recognition molecules containing amino groups were introduced into the microchannels and incubated for 1 hour to obtain a fluid microchannel interface containing recognition molecules, and the chip was placed in a refrigerator at 4° C. for use. After chip preparation, the chip was washed 3 times with PBS buffer, 100 μl each time, and secondary antibodies containing 20 μg/ml with fluorescence or 10 μM complementary strand of nucleic acid aptamers with fluorescence were introduced, incubated for 30 minutes, and then the chip was washed 3 times with PBS buffer, 100 μl each time, and imaged using an inverted fluorescence microscope (Nikon inverted fluorescence microscope), and when the ratio of the fluorescence value of the modified positive chip to that of the unmodified negative chip was greater than or equal to 1.5, the successful modification of the recognition molecule was demonstrated.
[0115] Recognition molecule coupling method II: The recognition molecules were attached to the microchannels by biotin-streptavidin interaction as follows: the microchannels of this specific example was immediately adhered to the carrier slide after irradiation through a plasma cleaner, and 20 μL ethanol solution of (3-mercaptopropyl)trimethoxysilane (MPTS) with a volume ratio of 4% was introduced every 5 minutes for 1 h. The operation can be performed by manual syringe injection or by automatic injection using an automatic microsampler, preferably Harvard Apparatus Pump 11 Pico Plus Elite syringe pump, US. Immediately afterwards, the channels were washed 3 times with ethanol solution, 100 μl each time, and placed in an oven for one hour at a heating temperature of 100° C. The chip was taken out, cooled to room temperature and 0.01% (mass fraction) of aromatic disulfide polyethylene glycol succinimidyl valerate, preferably o-Pyridyl disulfide polyethylene glycol succinimide valerate (with the polyethylene glycol molecular weight of 2000) was introduced.
[0116] After 30 minutes of reaction, the microchannels were rinsed with deionized water and 3 times with PBS buffer, 100 μl each time, and then 15 μg/ml of streptavidin were introduced into the microchannels and incubated for 1 hour to obtain a fluid microchannel interface modified with streptavidin, and the chip was placed in a refrigerator at 4° C. for use. One hour before use, the chip was taken out and rinsed 3 times with PBS buffer, 100 μl each time, then 20 μg/ml of recognition molecule containing biotin (human EpCAM/TROP1 biotinylated purified PAb, item number BAF960) was introduced into the microchannels and incubated for 1 hour to obtain a fluidic microchannel containing the recognition molecules. A schematic diagram of the coupling steps can be seen in
[0117] In the following, the effect of capture/release of fetal cells were tested using the chip made by Coupling Method II.
Example 3 Testing of the Capture Efficiency of Microfluidic Chip on Fetal Cells
3.1 Experimental Method
[0118] The microfluidic chip obtained by the recognition molecular coupling method II in Example 2 was taken and the chip was blocked for 30 minutes using 3% bovine serum albumin solution as the blocking solution.
[0119] An additional culturable cell line was added to 1 mL of healthy human peripheral blood to simulate the peripheral blood environment of a pregnant woman (the culturable fetal cell line was JEG-3 (human choriocarcinoma cell line), purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number TCHul95). Specifically, the blood contained approximately 100 JEG-3 cells, 9.63×10.sup.6 leukocytes per mL and 3.98×10.sup.9 erythrocytes per mL. The obtained blood was introduced by a syringe pump through the inlet (1), while the inlet (2) was simultaneously fed with buffer, at a flow rate of 0.3 mL/h. After the blood was completely introduced, the chip was rinsed with PBS buffer at a flow rate of 1.0 mL/h for 15 minutes. The JEG-3 cell line was pre-stained with calcein before being added to the blood to differentiate target cells from blood cells.
[0120] The staining method was: the cells were digested for 10 minutes using 0.02% EDTA (disodium EDTA) digestion solution at pH between 7.2 and 7.4, then the digestion solution was removed, and PBS buffer was added, and the cells were blown down to obtain a concentration of 1*10.sup.5 cells/ml. 200 μl of cell solution was added 1 μl of calcein solution (Thermo Fisher, item number C34852), stained at 37° C. for 10 minutes, and washed 3 times with 500 μl of PBS buffer and centrifuged at 1000 g for 3 minutes each time to obtain pre-stained cell solution. A schematic diagram of the capture steps can be seen in
[0121] The chips were washed and imaged by inverted fluorescence microscopy (excitation by blue laser). The number of fluorescent cells was counted to examine the capture efficiency of fetal cells, and the results are shown in Table 2.
[0122] Wherein, human B lymphocytoma cells Ramos and leukocyte WBCs were used as negative cells to examine the effect of specific recognition as well as non-specific adsorption of the chip. The operation was performed with reference to the treatment for JEG-3 cells.
3.2 Experimental Results
[0123]
TABLE-US-00002 TABLE 2 Analysis of capture efficiency Human B Simulated Lymphocytoma Cell Type Fetal Cells Cells Ramos Leukocytes Capture 92.3 ± 1.2% 3.2 ± 2.0% 0.08 ± 0.006% Efficiency
3.3 Comparative Example 2
[0124] Effect of Blood Pretreatment on Cells Capture
[0125] 100 JEG-3 cells were added to 1 mL of human peripheral whole blood to simulate the peripheral blood of pregnant women. The treatment of JEG-3 was performed with reference to the experimental method described in 2.1. 3 different methods were used for comparative analysis: 1) the whole blood was directly introduced into the chip for injection analysis without any treatment; 2) the peripheral blood and the prepared gradient separation solution (percoll density used 1.090) in a 4:3 volume ratio were taken into a centrifuge tube and centrifuged at 400 g for 30 min at 18-20° C. After centrifugation, four layers of solution were obtained, which are mature erythrocytes, centrifugal liquid layer, monocyte layer, platelets and plasma layer, and the monocyte layer was removed with a pasteur pipette, transferred to a 2 mL centrifuge tube, centrifuged, washed, and finally resuspended in 300 μL PBS for later use; 3) a erythrocyte lysate (potassium bicarbonate (KHCO.sub.3) 1.0 g; ammonia chloride (NH.sub.4Cl) 8.3 g; EDTA-Na.sub.2 0.037 g, double distilled water was added to 1000 mL, the erythrocyte lysis solution was then obtained) was taken, 5 volumes of which were mixed upside down with blood for 5 minutes, and the lysed solution was centrifuged at 400-500 g for 5 min, the red supernatant was discarded, and centrifuged at 4° C. for better effect to enrich the bottom mononuclear cells, and resuspended in 300 μL PBS for later use.
[0126] The samples treated by the above three methods were introduced into the microfluidic chip using the same method as in 3.1, with a flow rate of 0.3 mL/h, respectively. After washing, the chip was imaged by inverted fluorescence microscopy (excitation by blue laser), and the number of fluorescent cells was counted to examine the capture efficiency of fetal cells. The cell counts before and after capture by microfluidic chip under different peripheral blood treatment methods are shown in Table 3 and Table 4, respectively.
TABLE-US-00003 TABLE 3 Cell counts before capture by chip under different peripheral blood treatment methods Method of Treating Leukocytes/ Erythrocytes/ Target Example Peripheral Blood mL mL cells Example 3.1 Untreated Blood 9.63 × 10.sup.6 3.98 × 10.sup.9 200 ± 3 Comparative Percoll 1.090 7.03 × 10.sup.6 0.05 × 10.sup.9 120 ± 20 Example 2 Lysis of 2.61 × 10.sup.6 0.07 × 10.sup.9 70 ± 25 Erythrocytes
[0127] Although pre-treatment of blood can remove some red blood cells and serum, and reduce the complexity of the cell sample, it also causes variable cell loss.
[0128] In contrast, the technical solution of the present application allows for the running of whole blood. Thus, the loss of cells can be further reduced with few cells to be captured.
TABLE-US-00004 TABLE 4 Cell counts after capture by chip under different peripheral blood treatment methods Method of Treating Target Example Peripheral Blood Leukocytes Cells Example 3.1 Untreated Blood ~2000 ~180 Percoll 1.090 ~1500 ~108 Comparative Lysis of ~1000 ~63 Example 2 Erythrocytes Note: Since erythrocytes do not contain nuclei and will not affect subsequent analysis, they are not included in the statistical analysis.
Example 4 Assaying of the Release Efficiency of Fetal Cells
4.1 Experimental Method
[0129] 1) The fetal cells captured in Example 3 were released using the chemical cleavage method, the specific process are as follows: a solution of 50 mM dithiothreitol was introduced into the chip that had captured cells (inlet 1), which was incubated at 37° C. for 10 min, and then rinsed using PBS buffer containing 3% bovine serum albumin at a flow rate of 3 mL/h, with a total volume used of 1 mL, and the released cell suspension was collected through the outlet (3).
[0130] 2) The release efficiency was calculated by comparing the number of cells in the chip before and after release. The recovery rate of cells after release was counted by counting the number of cells in the final 1 ml of cell suspension. The reason for the difference between the release efficiency and the recovery rate may originate from the adsorption of cells by the channel during the collection process. This can be somewhat improved by blocking the chip with a blocking solution, which is a 3% bovine serum albumin solution. The number of cells was counted according to 3.1 Experimental Methods. A schematic diagram of the release steps can be seen in
[0131] (3) Viability analysis of the cells after release: the conventional double staining method (i.e. Calcein-AM (Calcein-AM) and propidium iodide (PI) solution) was used to analyze the viability of released cells. The specific method is described as follows: the recovered suspension of post-release cells was taken and centrifuged at centrifugal concentration, 1000 g for 3 min, to obtain 200 μl of suspension. 1 μl of Calcein-AM (Thermo Fisher, No. C3099) and 1 μl of PI solution (Sigma, No. P4864) were added and incubated for 30 min at 37° C. Imaging analysis was performed using a fluorescent inverted microscope. The number of live cells was obtained using filter Ex 465-495 nm/BA 521-558 nm imaging, and the number of cells was obtained using filter Ex 520-555 nm/BA 570-630 nm imaging. Cell viability was calculated as Cell viability=number of live cells/(number of live cells+number of dead cells)*100.
(4) Comparative Example 3
[0132] Trypsin digestion was used to release the fetal cells captured in Example 3 by introducing a 0.25% trypsin solution (Thermo Fisher, item number 25200056) into the chip that had captured cells (inlet 1), which was then incubating for 3 minutes at 37° C., and rinsing using PBS buffer containing 3% bovine serum albumin at a flow rate of 3 mL/h, with a total volume of 1 mL. The released cell suspension was collected through the outlet (3). Subsequent cell viability analysis experiments were performed with reference to experiment 4.1 (3).
4.2 Experimental Results
[0133] The visual graphical reference of the release is shown in
TABLE-US-00005 TABLE 5 Analysis of cell release efficiency, recovery rate and post-release viability Release Recovery Purity prior Purity after Example Efficiency Efficiency to Release Release Cell Viability Example 4.1 93.47% ± 6.99 73.34% ± 3.49 52.23% ± 4.84 86.46% ± 3.86 92.75% ± 0.16 Comparative 79.75% ± 4.16 50.56% ± 5.23 50.43% ± 4.04 55.23% ± 5.45 63.6% ± 4.34 Example 3
Example 5 Genome and Transcriptome Analysis of Captured Cells
5.1 Experimental Method
[0134] 1) Genomic analysis: Fetal cells released from Example 4 were collected in a 1.5 ml RNase-free Eppendorf tube and the supernatant was removed by centrifugation (1000 g for 3 min). The volume was concentrated to 10 μl and DNA was recovered using a DNA extraction kit or by thermal lysis. Thermal lysis was used for lysis in this example. Comparative experiments showed that the lowest DNA loss rate was obtained with the thermal lysis treatment. Samples were placed on a 95° C. heater, thermally lysed for 10 minutes, and then placed in a −80° C. refrigerator for storage (note: samples were stored for no more than 48 hours). DNA samples were tested using Bio-Rad's PrimePCR ddPCR assay kit, which detects the EGFR L858 mutation (Item #1863104; EGFR L858 mutation was detected here because the mutation occurred in the cells that were admixed, whereas normal blood samples were wild-type, as a way to perform specific analysis of the genetic analysis of the enriched cells). Data were analyzed using the Bio-Rad companion package to calculate the number of EGFR L858 mutations detected from a single sample.
[0135] 2) Transcriptome analysis: Fetal cells released from Example 4 were collected in a 1.5 ml RNase-free Eppendorf tube, the supernatant was removed by centrifugation (1000 g for 3 min). The volume was concentrated to 10 μl, and RNA recovery was performed with an RNA extraction kit. In this example, Zymo Research Corp's TRI Reagent (Item No. R2050-1-50) was used for cell lysis, and the experimental method was referred to the instructions. The collected RNA was purified using Zymo Research Corp's Direct-zol RNA MicroPrep (Item No. R2060) kit, and the experimental method was referred to the instructions. After obtaining the RNA, reverse transcription kits were used to reverse transcribe the RNA into cDNA. cDNA was reverse transcribed from the purified RNA using the Scientific Maxima H Minus (Item M1661) Reverse Transcriptase Kit from Thermo Fisher. The cDNA samples were tested using Bio-Rad's PrimePCR ddPCR assay kit, which covered 14 ROS1 gene rearrangement isoforms (item number qHsaCID0016464; the ROS1 gene was tested here because of the high expression of the adulterated cells and the absence or low expression of the normal blood). Data were analyzed using the Bio-Rad companion software package to calculate the corresponding copy number of ROS1 rearrangements detected from individual samples.
[0136] 3) Comparative Example 4, the method of lysis in the chip is briefly described: the microfluidic chip that had captured cells obtained in Example 3 was recovered for DNA or RNA. The recovery method was performed using the recovery kits described in steps 1) and 2) of Example 5.1 above. The corresponding reagents were introduced directly into the chip for cell lysis, and then the solution was aspirated and further purified.
5.2 Experimental Results
[0137] 1) For the detection of gene mutations in rare cells, the release method of the invention is superior to the method of direct cell lysis on a chip. The method of the invention can maintain a higher gene concentration, which in turn improves the detection rate and reduces the false negative rate. As shown in Table 5 below, even if the number of cells was as low as 2, the chip of the application can still obtain a better detection rate. In contrast, the detection rate was very low even when the number of cells was as high as 25 in Comparative Example 4.
TABLE-US-00006 TABLE 6 Detection rate ratio analysis for mutant/wild type 2 cells 13 cells 25 cells Example captured captured captured Example 5.1 46.87% ± 1.96 49.43% ± 2.55 65.43% ± 3.96 Comparative 1.61% ± 0.17 1.60% ± 0.10 12.37% ± 0.80 Example 4
[0138] 2) The results of detection of the cellular transcriptome are shown in Table 7. The release method of the invention is superior to the method of cell lysis directly on the chip, maintaining a higher gene expression analysis, which in turn improves the detection rate and reduces the false negative rate.
TABLE-US-00007 TABLE 7 Copy number analysis for transcriptome ROS1 Example ROS1 Copy number (k) Example 5.1 16.37 ± 1.06 Comparative 8.40 ± 0.79 Example 4
Example 6 Whole Genome Analysis for Fetal Cells
6.1 Experimental Method
[0139] 1) For the fetal cells released in Example 4, picking of fetal cells was achieved by fluorescence microscopy. The obtained cells were collected in a 0.2 ml of RNase-free Eppendorf tube. The transfer reagent used was controlled to 1 μl or less. Follow-up operations were performed using a commercial whole genome amplification kit, MALBAC® Single Cell Whole Genome Amplification Kit (Shanghai Yikon Medical Laboratory Co., Ltd.). After cell amplification, the product was purified using magnetic beads (for example, Nanjing Vazyme Biotechnology Co., Ltd., item No. N412-01). Whole-genome library was established on the obtained genomes (e.g., Nanjing Vazyme Biotechnology Co., Ltd., item No. TD502-01), and the products were purified using magnetic beads (e.g., Nanjing Vazyme Biotechnology Co., Ltd., item No. N412-01). After quantification, the products were sent to the testing company for sequencing analysis (e.g., Beijing Novogene Technology Co., Ltd, second generation whole genome sequencing analysis, NovaSeq technology (Illumina)). The whole genome copy number of fetal cells was obtained by comparison with the reference sequence number h19.
[0140] 2) Reference Example
[0141] In order to verify the genomic integrity of the obtained cells, the original cell solution without any chip treatment and release was used as a reference for sequencing analysis and comparison with the results of Example 6.1 Step 1). The raw cell solution was processed as follows: 10.sup.5 cells were taken and genes were extracted using the Genome Extraction Kit (Nanjing Vazyme Biotechnology Co., Ltd., item no. DC111-01). Whole-genome library was established on the obtained genomes (Nanjing Vazyme Biotechnology Co., Ltd., No. TD501-01), and the products were purified using magnetic beads (Nanjing Vazyme Biotechnology Co., Ltd., No. N412-01). After quantification, the products were sent to the testing company for sequencing analysis (Beijing Novogene Technology Co., Ltd, second generation whole genome sequencing analysis, NovaSeq technology (Illumina)). The whole genome copy number of the original cell solution was obtained by comparing with the reference sequence number h19.
6.2 Analysis of Experimental Results
[0142] As shown in