CN111948391A

CN111948391A - Array sensor based on nano metal organic framework for histological diagnosis of colon cancer

Info

Publication number: CN111948391A
Application number: CN201910416356.6A
Authority: CN
Inventors: 李根喜; 吴帅; 韩祎巍; 孙召伟; 王琳
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2020-11-17

Abstract

The invention discloses a method for constructing novel array sensing for colon cancer histological diagnosis based on a nano metal organic framework, provides a novel method for human tumor classification, and provides a novel idea for the application of MOF in biosensing. We selected three structurally stable NMOF (Cu-MOF, Fe-MOF and Zr-MOF) and two 5-TAMRA labeled single stranded DNAs, and designed six NMOFs-DNA complexes as recognition elements (NP1-NP 6). The fluorescence of the DNA in the compound is quenched by the nanoparticles, when the tissue lysate of the sample is incubated with the recognition element, the protein interacts with the NMOF surface through a plurality of weak interaction forces, selectively replaces the DNA molecules serving as the report probe and generates the change of the fluorescence intensity to form a sensing array, and different tumor tissues can be effectively recognized. The method provides a high-discrimination and strong-adaptability alternative scheme for identifying the tumor tissue of the colon cancer, and has wide development prospect in medical diagnosis.

Description

Array sensor based on nano metal organic framework for histological diagnosis of colon cancer

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a construction principle, a process, experimental conditions and application of a novel array sensing system based on a nano metal organic framework.

Background

Tissue biopsy, as a test method for accurately diagnosing cancer, is a typical complicated sample analysis process, mainly involving the extraction of cells or tissues and a diagnosis process for identifying the occurrence and degree of cancer. Colon cancer is one of the most common malignancies in the world and is also the fourth largest type of cancer causing death. While differentiation of cancer tissue from normal colonic epithelial cells is primarily due to differences in protein expression. Histopathological examination by microscopic observation including immunohistochemistry and enzyme histochemistry are the gold criteria for diagnosis of colon cancer. However, there are no specific single or multiple tumor markers in colon tissue as a valid criterion to distinguish between normal and tumor tissue. In addition, the process of paraffin embedding and tissue sectioning requires trained laboratory operators, and medical results depend on the individual judgment of the clinician, and such differences in subjectivity are prone to inaccurate diagnosis. Therefore, there is an urgent need to design a rational histological diagnosis method for colon cancer.

The array recognition method can provide a selective strategy for the sensory analysis of complex samples, which takes the specific target and receptor binding recognition event as the basis. Nanoscale Metal Organic Frameworks (NMOFs) are an emerging molecular nanomaterial and are ideal elements for designing various artificial receptors due to the adjustability and versatility of synthesis. The high selectivity of the metal-containing units and the organic ligands gives a diversity of species of NMOFs, such as the MIL or UiO series. In addition, NMOFs have a suitable surface area, very useful for binding biomolecules. Typically, it has been shown that labelled single stranded DNA (ssdna) can be efficiently adsorbed on the surface of NMOF, providing higher selectivity for control of the sequence and length of the DNA. The diversity of surface charges and aromatic groups leads to different degrees of interaction with biomolecules, which allows for easy design of various receptors for array recognition sensing. In view of the multifunctional design and smart DNA structure of NMOF, we speculate that the assembly of both might be expected to construct a sensor array with excellent performance for bioanalysis of complex samples.

Disclosure of Invention

The invention aims to construct an array sensing system and provide a method for improving the accuracy of colon cancer histological diagnosis and shortening the examination time.

The invention principle is as follows:

based on the selective non-covalent interaction between colon tissue components and DNA adsorbed NMOFs, a fluorescence non-specific recognition system based on array sensing is constructed. Three NMOFs were used as core moieties for artificial receptors and as nanoquenchers for fluorescently labeled DNA sequences. When interacting with various analytes, a plurality of intermolecular forces such as electrostatic interaction, coordination interaction, pi stacking and hydrogen bond attraction are formed at reactive organic parts or accessible coordination sites on the surface of the NMOF, and fluorescence intensity changes are caused by pi stacking and hydrogen bond attraction, and a fingerprint is directly drawn by a Linear Discriminant Analysis (LDA) method, so that sensitive identification and analysis are performed on a protein target. Using these elements, cancer colon cell lines and tissues can be effectively distinguished from non-cancer cell lines and tissues due to the unique proteomic characteristics within the cell. The whole translation process greatly shortens the inspection time and improves the efficiency. In addition, it has been successfully used to identify 42 clinical samples and has a high degree of accuracy, enabling preliminary clinical applications. In general, this approach provides a new approach to human tumor classification and broadens the application of MOFs in biosensing.

The required reagents:

the sequence of the Azo-DNA oligonucleotide synthesized by Shanghai Biotechnology Ltd (Shanghai, China) is: 5 '- (CHO) -CCTAGCAACAGACCGCACTTTATGATAGCAA(Azo) GC (azo) TA (azo) GG-3'. PAA is purchased from Zhaoyu element nanotechnology GmbH (China Anhui). (3-aminopropyl) triethoxysilane (APTES), potassium chloride (KCl) and other analytical reagents were purchased from Sigma-Aldrich (Shanghai, China) or Sigma-Aldrich Co., Ltd (St Louis, Mo., USA). All solutions were prepared with deionized water and purified using a Milli-Q purification system (Bedford, MA, USA) to a resistance of 18.2 M.OMEGA.cm.

The preparation method comprises the following steps:

firstly, synthesizing nanoscale MOF:

Cu-MOF: 50mL of an aqueous methanol solution containing Cu (NO) was stirred at room temperature₃)₂·3H₂O (0.9g) and PVP (0.4 g). Then H is introduced₃BTC (0.43g) was dissolved in another 50mL of methanol to form a ligand mother liquor. The solution was then added dropwise to the metal precursor liquid using a syringe. The formation of a blue colloidal suspension was observed during the dropping process for about 10 minutes, and then the stirring was stopped, and the mixture was allowed to stand for 24 hours to allow stable growth of colloidal nanoparticles under a dark condition. The resulting blue solid was centrifuged at 10000rpm for 10min and washed several times with fresh methanol. Finally, the precipitate was dried under vacuum at 60 ℃ overnight for later use.

Fe-MOF：FeCl₃·6H₂A solution of O (1.350g) and BDC (0.412g) in 30mL DMF was ultrasonically dissolved in a 50mL volume of a tetrafluoroethylene-lined hydrothermal kettle and heated at 110 deg.C for 20 hours. The resulting orange solid was harvested by centrifugation at 10500rpm for 10 minutes and washed several times with fresh ethanol. Finally, the precipitate was dried under reduced pressure at 120 ℃ for 6 hours under vacuum.

Zr-MOF: reacting ZrCl₄(37.5mg) and TCPP (6.5mg) were dissolved in 16.25mL of DMF and sonicated for about 3 minutes. Dichloroacetic acid (0.25mL) was then added to the solution and the mixture was charged to a tetrafluoroethylene lined hydrothermal kettle and heated at 130 ℃ for 18 hours. The dark purple solid was collected by centrifugation at 10500rpm for 10 minutes and washed several times with fresh DMF and ethanol. Finally, the precipitate was dried under vacuum at 130 ℃ overnight for later use.

Secondly, sequence design:

DNA 1：AAAAAA AAAAAAAAAAAA

DNA 2：CCCCCCCCCCCCCCCCCCCC

thirdly, cell culture and treatment:

NCM-460 and Colo 205 cell lines were cultured in RPMI 1640 medium (Gibco, Invitrogen) containing 10% FBS and placed in 5% CO₂Cultured in an incubator (Thermo 3111). HCT-116 at McCoy's 5A medium (Gibco, Invitrogen) and the other culture conditions were the same as described above. Cells were harvested by centrifugation at 1000rpm for 5 minutes and washed twice with PBS buffer. The resulting cells were resuspended in PBS solution to form a cell suspension. The numbers were counted using an automated cell counter (Invitrogen counter) for later use.

Fourthly, acquiring and treating colon cancer tissues:

all patients were organized from the second subsidiary hospital of the southeast university. All cases received informed consent and the study was approved by the scientific ethics committee of the university of Nanjing and the university of southeast.

Histopathological examination: the obtained tissues were dehydrated and embedded in paraffin, and then sectioned, followed by H & E staining. The observation was performed using a microscope while recording the image.

Preparation of lysate: first, fresh tissue was washed twice with cold PBS buffer. Precooled extraction reagent (one-step method protein extraction kit active in animal tissue, bio-biotechnology limited) was then added to the collected tissue and sonicated (30 seconds each, 3-4 times each, 1 minute each). After sonication, the tissue lysates were centrifuged at 12000rpm (4 ℃) for 10 minutes and the supernatants were transferred to new EP tubes for protein quantification and subsequent sensing experiments.

Fifthly, a sensing analysis experiment:

for the sensing study of interest, two fluorescently labeled DNA strands (DNA 1 and 2) were incubated with three NMOFs respectively and diluted with HEPES buffer (10mM, 100mM NaCl, pH 7.2) such that the final concentrations of DNA element, Cu-MOF, Fe-MOF and Zr-MOF were 50nM, 1. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL, respectively. The mixture (200 μ L per well) was transferred to a black 96-well plate (Corining inc., America). After 30 minutes of incubation, the fluorescence intensity at 570nm was measured on a multifunctional microplate at an excitation wavelength of 540nm (I)₀). When the target of the assay (protein, cell line or tissue lysate of colon cancer) was introduced and incubated for 30 minutes (cell line 1 hour), the reaction system of each well gave a different response (I) at 570 nm. The difference between the two values (Δ I ═ I-I)₀) As matricesThe response signal of the column sensing analysis. Each set of target experiments was repeated six times. Finally, the raw data matrix was processed using Xlstat (2015 edition) and Linear Discriminant Analysis (LDA) in IBM SPSS Statistics 24 software.

Eight, conclusion

In summary, we have demonstrated that NMOF modules with DNA elements can make fluorescent sensor arrays capable of analyzing tumor tissue for colon cancer and normal tissue. NMOF size and surface morphology provide selective interaction with proteins, and the intrinsic quenching fluorescent properties facilitate signal transduction of binding events. Using a sensor array, we can rapidly (1 hour) identify a variety of analytes, ranging from pure standard samples to complex clinical samples, with high sensitivity and accuracy by LDA analysis. The short-time detection greatly improves the diagnosis efficiency. Notably, complete differentiation was achieved to analyze different tissue samples for colon cancer, at levels as low as 150ng, thereby minimizing biopsy tissue size. In addition, the method has already carried out clinical preliminary diagnosis on unknown colon cancer tissues, and provides a supplementary strategy for the traditional histological examination method. In summary, we consider the NMOF-based sensor array to have broad prospects in biomedical diagnostics.

The principle of the device, the method of implementation, the detection conditions, etc. of the present invention have been described in detail above, but the present invention is not limited to the above-mentioned specific details of detection, and the present invention can be modified to the detection substrate within the technical idea of the present invention, and the modification of the detection substrate is within the protection scope of the present invention.

Drawings

FIG. 1: an array sensor structure and a schematic diagram based on a nano metal organic framework. (A) The structural types of the three NMOFs used in this study; (B) using nano-assembly between NMOFs and two single stranded DNAs, 6 recognition elements (NP1-NP6) were designed. The prepared sensor array produces different responses to different binding processes.

FIG. 2: PXRD atlas and TEM image characterization of three NMOFs, respectively (a and b) Cu-MOFs, (c and d) Fe-MOFs, (e and f) Zr-MOFs.

FIG. 3: fluorotitrating fluorescently labeled DNA 1 at 570nm against Fe-MOFs, the NP3 recognition element. The fluorescence intensity was normalized and plotted against the mass concentration of Fe-MOFs. The DNA concentration was 50 nM. The inset shows the change in fluorescence intensity with the addition of a concentration gradient of Fe-MOFs under UV light.

FIG. 4: protein was analyzed at a concentration of 5 μ M based on array sensing. (A) Fluorescent response of the sensing array (NP1-NP6) to multiple proteins: bovine Serum Albumin (BSA), hemoglobin (Hb), lysozyme (Lyso), horseradish peroxidase (HRP) and proteinase K, error bars represent standard deviations of six parallel measurements; (B) heatmaps from the fluorescence response patterns of the five proteins; (C) typical score plots of the first two standard factors of the fluorescence response pattern obtained by LDA analysis of five proteins at 5 concentrations of 5 μ M, with 95% confidence ellipses.

FIG. 5: array-based cell line sensing. (ii) fluorescence response (. DELTA.I, I-I)₀) Patterned sensor arrays (NP1-NP6) for various constant cell number (. about.1000) cell lines (NCM 460, Colo 205, and HCT 116); (B) the LDA analysis obtained typical score plots of the first two factors of the fluorescence response pattern of the three cell lines, 95% confidence ellipses.

FIG. 6: light (a) medical results predicted with a sensing array were compared to gold standards for histopathological sections; (B) each distributed point represents 42 samples based on a standard distribution graph obtained by the established data model; (C) performing LDA analysis using only two sets of samples of cancer and normal individuals, the LDA dataset having been converted to a statistical histogram; (D) the ROC analysis was performed on both sets of samples and the area under the curve AUC value was 0.90.

Claims

1. Claim 1 claims the idea of the inventive concept: three NMOF and two single-stranded DNA labeled by 5-TAMRA construct 6 complex recognition elements (NP1-NP6), and form a sensing array aiming at different target proteins to carry out differential analysis on tumor tissues. Three NMOF (Cu-MOF, Fe-MOF and Zr-MOF) with stable structures are selected, and respectively contain metal ions with different valence states and organic ligands with different carboxyl numbers, and the three NMOF (Cu-MOF, Fe-MOF and Zr-MOF) are used as a scaffold for constructing a chemical nose/tongue probe. As shown in scheme 1, we designed six recognition elements (NP1-NP6) and fabricated sensor arrays by nano-assembly using these NMOF and two 5-TAMRA labeled single stranded DNAs, respectively rich in adenine and cytosine. Due to the high plasticity of the DNA structure, the non-covalent NMOFs-DNA complex (where the fluorescence of the DNA is quenched by the nanoparticles) has highly controllable binding properties. While when the corresponding tissue lysates are incubated with these complexes, the proteins interact with the NMOF surface through a variety of weak interaction forces (e.g., electrostatic, hydrogen bonding interactions and hydrophobic interactions), resulting in different competitive or synergistic interfacial behavior of the DNA molecules. The DNA molecules serving as the report probes are selectively replaced, the change of fluorescence intensity is generated, a sensor array is formed by the collected signals from the target receptor pair, and a fingerprint with distinguishing capability is drawn, so that different tumor tissues can be effectively identified. The method provides a new method for human tumor classification, and widens the application of MOF in biosensing.

2. Claim 2 claims protection of the array sensor based on the nano metal organic framework comprising the following steps: (1) a complex structure formed by NMOF and DNA; (2) synergistic competition of proteins with NMOF-DNA; (3) protein identification is used for histological diagnosis of colon cancer.

3. Claim 3 claims the design of the NMOF-DNA complex in this patent: NP1-NP 6.