Polypeptide probe and kit for early diagnosis of huntington's disease

Abstract

A polypeptide probe specifically binding to mutant huntingtin, a polypeptide probe specifically binding to neurofilament light, a kit and a microfluidic chip containing the polypeptide probe, a method for preparing the polypeptide probe, and the use of the polypeptide probe.

Description

Peptide probes and kits for early diagnosis of Huntington's disease Technical Field

[0001] The present disclosure relates to the field of biomedical technology, and in particular to a polypeptide probe that specifically binds to a mutant huntingtin protein, a polypeptide probe that specifically binds to a neurofilament light chain, a kit and a microfluidic chip comprising the polypeptide probe, a method for preparing the polypeptide probe, and applications of the polypeptide probe. Background Art

[0002] Huntington's disease (HD) is a familial, inherited neurodegenerative disorder characterized clinically by movement disorders, mental disorders, and dementia. The global prevalence of HD is approximately 2.7 per 100,000 individuals, with an annual incidence of approximately 0.38 per 100,000 individuals. The disease peaks between the ages of 30 and 50, and the average life expectancy is approximately 15 to 20 years. The disease is associated with high disability and mortality rates, with patients gradually losing the ability to care for themselves. Currently, there is no specific treatment, and a cure is unavailable. The gene encoding the abnormal huntingtin protein is inherited in a dominant manner; if a parent has HD, their children have a 50% chance of inheriting the disease. HD typically develops after age 40, and early symptoms can be easily mistaken for epilepsy or depression. Therefore, by the time patients discover the disease, the disease-causing gene has often already been passed on to their offspring. Electroencephalograms (EEGs) and imaging tests, currently commonly used diagnostic methods, are nonspecific and therefore ineffective for early diagnosis. To effectively prevent the occurrence of the disease, early genetic diagnosis of Huntington's disease is of great significance. Huntington's disease is caused by excessive repetitions (>35) of the CAG trinucleotide sequence in the first exon of the Huntington gene, encoding the mutant huntingtin protein (mHtt).

[0003] Neurofilaments (NF) are essential components of the neuronal cytoskeleton, maintaining normal neuronal morphology and nerve fiber elasticity to prevent breakage. Neurofilament light chain (NFL) is considered the most important component of NF. NF is a neuronal cytoskeletal protein highly expressed in large-diameter myelinated axons. When central nervous system axons are injured by conditions such as inflammation, neurodegeneration, trauma, or cerebrovascular disease, NF is released into the extracellular fluid, cerebrospinal fluid, and, at lower concentrations, into the peripheral blood. The amount released depends on the severity of the axonal injury. In recent years, NFL has been garnering increasing attention as a candidate biomarker for axonal injury. With the rapid advancement of technology, the detection of NFL has become more precise. Clinical studies have shown a close correlation between NFL and various diseases, offering important insights into disease diagnosis, prognosis, biomarker development, disease severity estimation, drug efficacy testing, and prognostic prediction. Research has shown that NFL, as a marker of axonal damage, plays an important role in the diagnosis, medication efficacy, and prognosis of various neurological diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), traumatic brain injury (TBI), Huntington's disease (HD), and acute ischemic stroke (AIS). Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. AD patients typically experience changes in memory, judgment, behavior, and mood, ultimately interfering with their ability to control their body. Due to an aging population and improved quality of life, the number of people with AD is expected to increase severalfold over the next few decades, from approximately 50 million worldwide today to over 100 million by 2050. Alzheimer's disease not only causes disability and immense suffering for patients, but also creates a significant financial burden. Furthermore, the incidence of Alzheimer's disease increases with age. According to epidemiological data, the prevalence is approximately 5% in those aged 65 and older, rising to 10% in those over 70, and 30% in those over 80. Currently, there are no effective treatments for Alzheimer's disease. Existing medications can improve symptoms in some patients, but they are expensive, have limited efficacy, and cannot halt or reverse the progression of the disease. As the population ages, while awaiting much-needed drug breakthroughs, attention is being focused on secondary prevention measures such as early identification and treatment of Alzheimer's disease.There is an urgent need for ideal AD diagnostic technologies that are highly accurate and reliable, non-invasive or minimally invasive, cost-effective, and can be used for large-scale screening to improve pathological diagnosis, achieve early diagnosis, and increase the possibility of treatment.

[0004] As a biomarker of axonal injury, accurate measurement of NFL concentrations can help understand the progression of various diseases. Central nervous system axonal damage caused by various disease states releases NFL into the extracellular fluid, cerebrospinal fluid, and peripheral blood. Peripheral blood levels are lower than those in the extracellular fluid and cerebrospinal fluid. In clinical practice, sampling cerebrospinal fluid is invasive and less readily available than peripheral blood. Therefore, accurate methods for detecting NFL in peripheral blood are highly desirable to guide clinical practice. Therefore, measuring NFL concentrations in body fluids, particularly readily accessible blood, holds promise for preventive screening in the preclinical stages of neurological diseases. However, compared to cerebrospinal fluid, blood is more complex and contains lower levels of biomarkers, necessitating the development of more sensitive and specific blood assays. Serum neurofilament light chain protein is a blood marker that reflects the extent of axonal damage and can aid in diagnosis, disease severity assessment, and prognosis in many neurological diseases. Currently, studies on probes targeting NfL are limited, clinical data are insufficient, and specificity and sensitivity analysis for individual diseases is lacking.

[0005] The combined detection of mHTT and NfL can effectively achieve early diagnosis of Huntington's disease and provide a reliable basis for prenatal diagnosis and genetic counseling.

[0006] Biochemical detection of changes in relevant biomarkers can, to a certain extent, promptly reflect disease progression. Based on the detection method, these biomarkers can be categorized into imaging biomarkers, cerebrospinal fluid biomarkers, and blood biomarkers. Imaging biomarkers for AD primarily include MRI and PET, which reveal brain atrophy and aggregates of proteins such as Aβ and tau in the brain, respectively. However, the complexity and high cost of these techniques limit their application in primary care settings. Cerebrospinal fluid (CSF) is the most reliable and accurate biofluid for assessing AD biomarkers because CSF directly interacts with the extracellular space in the brain, reflecting relevant biochemical / pathological changes and thus being included in AD diagnostic criteria. However, the complex and invasive nature of CSF extraction limits the application of these methods in patients with Alzheimer's disease and mild cognitive impairment. Due to the routine and readily available nature of blood testing, which allows for sample collection and processing using existing systems, blood-based AD biomarkers can meet the requirements of widespread population screening in primary care settings. Confirming the cause of dementia through blood testing offers the advantages of cost-effectiveness, non-invasiveness, and ease of implementation, potentially making large-scale dementia diagnosis a reality. However, blood-based biomarkers have picogram concentrations and require ultrasensitive sensors and analytical techniques for detection.

[0007] Peptide probes have the characteristics of good selectivity, low immunogenicity, good biocompatibility, strong penetrability, and easy excretion and clearance. The construction of peptide probe libraries through the mixed splitting method can effectively increase the diversity of molecular probes. High-throughput screening methods can further screen for effective probes. Combining these two methods can produce high-affinity probes, thereby enabling early diagnosis of Huntington's disease. Microfluidic chips have the advantages of high throughput, fast analysis speed, low contamination, small sample volume, low cost, and safety. They have broad development prospects in the fields of clinical diagnosis and disease screening. Combining high-affinity small molecule probes with microfluidic chips can effectively improve the sensitivity and timeliness of detection, while further reducing the complexity and cost of the operation, and is the trend of future diagnosis.

[0008] Summary of the Invention

[0009] The present disclosure provides a polypeptide probe that specifically binds to mutant huntingtin protein, a polypeptide probe that specifically binds to neurofilament light chain, a kit and a microfluidic chip comprising the polypeptide probe, a method for preparing the polypeptide probe, and applications of the polypeptide probe.

[0010] According to one aspect of the present disclosure, a polypeptide probe that specifically binds to a mutant huntingtin protein is provided, which has the following structure from N-terminus to C-terminus: M-(AA1) n -N-(AA2) m, wherein M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, m<5.

[0011] In some embodiments, AA2 is Gln.

[0012] In some embodiments, the polypeptide probe is selected from the group consisting of:

[0013] mHBP265: (Asn-Gln-Phe-Gln-Gln-Gln);

[0014] mHBP1087: (Gln-Gln-His-Gln-Gln-Gln); and

[0015] mHBP3296: (Lys-Gln-Phe-Gln-Gln-Gln).

[0016] In some embodiments, AA1 is Gln.

[0017] In some embodiments, the polypeptide probe is selected from the group consisting of:

[0018] mHBPL1: Asn-Gln5-Phe-Gln-Gln-Gln;

[0019] mHBPL2: Gln-Gln5-His-Gln-Gln-Gln;

[0020] mHBPL3: Lys-Gln5-Phe-Gln-Gln-Gln;

[0021] mHBPL4:Lys-Gln 10 -Phe-Gln-Gln-Gln;

[0022] mHBPL5:Gln-Gln 10 -His-Gln-Gln-Gln; and

[0023] mHBPL6:Asn-Gln 10 -Phe-Gln-Gln-Gln.

[0024] In some embodiments, the present disclosure provides bivalent or multivalent assemblies of polypeptide probes that specifically bind to mutant huntingtin protein, wherein the bivalent or multivalent assemblies are formed by covalent linkage via linker molecules, or by mixing with multimers and non-covalent linkage.

[0025] According to another aspect of the present disclosure, a polypeptide probe specifically binding to neurofilament light chain is provided, which has the following structure from N-terminus to C-terminus: XY-(Ala)n -Z-Thr-Ser or XY-(Gly) n -Z-Thr-Ser, wherein the amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, and n is less than 10.

[0026] In some embodiments, XY is Met-Trp or Leu-Tyr.

[0027] In some embodiments, the polypeptide probe is selected from the group consisting of:

[0028] NBP596: Met-Trp-Ala-Gln-Thr-Ser;

[0029] NBP775: Met-Trp-Ala-Asn-Thr-Ser; and

[0030] NBP4061: Leu-Tyr-Ala-Asn-Thr-Ser.

[0031] In some embodiments, the polypeptide probe is selected from the group consisting of:

[0032] NBPL1: Leu-Tyr-Ala3-Asn-Thr-Ser;

[0033] NBPL2: Leu-Tyr-Ala5-Asn-Thr-Ser;

[0034] NBPL3:Leu-Tyr-Ala7-Asn-Thr-Ser; and

[0035] NBPL4:Leu-Tyr-Ala9-Asn-Thr-Ser.

[0036] In some embodiments, the present disclosure provides bivalents or multivalents formed by assembling polypeptide probes that specifically bind to neurofilament light chains, wherein the bivalents or multivalents are formed by covalently linking via a linker molecule, or by mixing with a multimer and non-covalently linking. According to another aspect of the present disclosure, a kit is provided, comprising at least one of the above-mentioned polypeptide probes that specifically bind to mutant huntingtin protein, bivalents or multivalents formed by assembling polypeptide probes that specifically bind to mutant huntingtin protein, polypeptide probes that specifically bind to neurofilament light chains, and bivalents or multivalents formed by assembling polypeptide probes that specifically bind to neurofilament light chains.

[0037] In some embodiments, the polypeptide probe is labeled with fluorescein or biotin.

[0038] In some embodiments, the polypeptide probe is labeled with rhodamine B.

[0039] In some embodiments, the kit further comprises a surfactant, a buffer, and EDTA.

[0040] In some embodiments, the surfactant is selected from one or a combination of Tween 20, sodium dodecyl sulfate (SDS), and the fatty acid sorbitan Span.

[0041] In some embodiments, the volume ratio of sodium dodecyl sulfate (SDS) and the fatty acid sorbitan Span is 1:1.5.

[0042] According to another aspect of the present disclosure, a microfluidic chip is provided, wherein a reaction zone thereof is coupled to at least one of the above-mentioned polypeptide probes that specifically bind to mutant huntingtin protein, bivalents or multivalents formed by assembling polypeptide probes that specifically bind to mutant huntingtin protein, polypeptide probes that specifically bind to neurofilament light chain, and bivalents or multivalents formed by assembling polypeptide probes that specifically bind to neurofilament light chain.

[0043] According to another aspect of the present disclosure, a method for preparing the above-mentioned polypeptide probe that specifically binds to mutant huntingtin protein is provided, the method comprising the following steps:

[0044] i) constructing a peptide probe library using a hybrid splitting method,

[0045] ii) Using magnetic beads for primary screening, peptide probes were enriched using mutant huntingtin protein.

[0046] iii) optimizing the polypeptide probe, comprising:

[0047] a) The polypeptide probe has the following structure from N-terminus to C-terminus:

[0048] M-(AA1) n -N-(AA2) m ,

[0049] Wherein, M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, and m<5.

[0050] b) Using molecular dynamics curves, the library is coarsely screened to obtain high-affinity candidate peptide probes.

[0051] c) Combined with the target protein structure, the common structural characteristics of candidate probes are captured by intermolecular forces such as hydrogen bonds and hydrophobic interactions. By combining the epitope sequence, the intermolecular forces related to the corresponding probe binding domain are amplified to further optimize the peptide probe structure.

[0052] According to another aspect of the present disclosure, a method for preparing a polypeptide probe that specifically binds to a neurofilament light chain is provided, the method comprising the following steps:

[0053] i) constructing a peptide probe library using a hybrid splitting method,

[0054] ii) Using magnetic beads for primary screening, the neurofilament light chain was used to enrich the peptide probes.

[0055] iii) optimizing the polypeptide probe, comprising:

[0056] a) The polypeptide probe has the following structure from N-terminus to C-terminus:

[0057] XY-(Ala) n -Z-Thr-Ser or XY-(Gly) n -Z-Thr-Ser,

[0058] The amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, and n is less than 10.

[0059] b) Using molecular dynamics curves, the library is coarsely screened to obtain high-affinity candidate peptide probes.

[0060] c) Considering steric hindrance and intermolecular forces, the distance between the hydrophilic end and the hydrophobic end is increased to further extend the N-terminal hydrophobic chain.

[0061] According to another aspect of the present disclosure, provided is the use of a polypeptide probe that specifically binds to a mutant huntingtin protein, or a bivalent or multivalent assembled from polypeptide probes that specifically bind to a mutant huntingtin protein, in the preparation of a therapeutic drug, diagnostic reagent, diagnostic kit, or imaging agent for Huntington's disease.

[0062] According to another aspect of the present disclosure, provided is the use of a polypeptide probe that specifically binds to a neurofilament light chain, or a bivalent or multivalent assembled from polypeptide probes that specifically bind to a neurofilament light chain, in the preparation of a drug, diagnostic reagent, diagnostic kit, or imaging agent for the prevention or treatment of a neurofilament light chain-related disease.

[0063] According to another aspect of the present disclosure, a method for detecting a sample using the above-mentioned microfluidic chip surface plasmon resonance imaging technology is provided, comprising:

[0064] Place the microfluidic chip on the prism;

[0065] Adjust the incident light angle so that the incident light is totally reflected at the junction of the chip and the prism;

[0066] Pass the sample into the chip;

[0067] Collect reflected light intensity;

[0068] Software is used to analyze changes in reflected light intensity to distinguish different samples.

[0069] The screening design scheme for preparing polypeptide probes disclosed in the present invention can quickly and effectively obtain high-affinity polypeptide probes. Specifically, in the method for screening probes disclosed in the present invention, the library construction of polypeptide probes by the mixed splitting method can effectively improve the diversity of molecular probes, and the effective probes can be further screened by the high-throughput screening method, and high-affinity polypeptide probes can be quickly and effectively obtained, thereby realizing the early diagnosis of Huntington's disease. The kit and microfluidic chip provided by the present invention have the advantages of high throughput, fast analysis speed, low pollution, small sample amount required, low cost, and safety, and have broad development prospects in the field of clinical diagnosis and disease screening. Combining high-affinity small molecule probes with microfluidic chips can effectively improve the sensitivity and timeliness of detection, while further reducing the complexity and cost of operation, which is the trend of future diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly describe the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0071] Figure 1 is a schematic diagram of the basic principle of probe primary screening.

[0072] FIG2 shows the results of detecting probes mHBP265, mHBP1087, and mHBP3296 using surface plasmon resonance imaging technology using mutant huntingtin protein.

[0073] FIG3 shows the results of detecting probes mHBPL1, mHBPL2, mHBPL3, mHBPL4, mHBPL5, and mHBPL6 using surface plasmon resonance imaging technology using mutant huntingtin protein.

[0074] FIG4 shows the results of detecting probes NBP596, NBP775 and NBP4061 using neurofilament light chain by surface plasmon resonance imaging technology.

[0075] FIG5 shows the results of detecting probes NBPL1, NBPL2, NBPL3, and NBPL4 using neurofilament light chain by surface plasmon resonance imaging technology.

[0076] FIG6 shows a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure.

[0077] FIG7 shows experimental data obtained by combining a polypeptide probe that specifically binds to mutant huntingtin protein and a polypeptide probe that specifically binds to neurofilament light chain for sample detection. DETAILED DESCRIPTION

[0078] The following will be combined with the accompanying drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the embodiments described are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0079] Unless otherwise defined, all terms (including technical and scientific terms) used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the relevant art and / or the context of this specification, and will not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0081] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions were followed. Materials, reagents, or instruments used in the following examples without manufacturer specified are all commercially available conventional products unless otherwise specified.

[0082] Unless otherwise specified, in the present disclosure, “nM” refers to “n mol / L,” “μM” refers to “μmol / L,” and “mM” refers to “m mol / L.”

[0083] The "mixing and splitting" method used in the embodiments of the present disclosure, which involves mixing and splitting resin beads after each reaction round, exhausts all possible synthetic products with a minimum number of synthetic steps.

[0084] In the disclosed embodiments, surface plasmon resonance imaging (SPRi) technology was used to characterize the performance of the synthesized probe. The basic principle of surface plasmon resonance imaging (SPRi) is as follows: With the help of a prism, a beam of parallel p-polarized light (most commonly single-wavelength light) is directed at a certain angle onto a free-electron-rich metal film assembled with different sample arrays. When the incident angle is greater than the critical angle for total internal reflection, the incident light resonates with the electromagnetic field generated by the free electron fluctuations on the metal film, namely surface plasmons (excitons), resulting in absorption. This absorption is related to the dielectric constant of the metal film and, therefore, to the sample array points loaded on the film surface. Different attached samples have different degrees of refractive index changes, resulting in different resonant absorption. As a result, the intensity of the (totally) reflected light received from the other side of the prism is inevitably different. When imaging with a charge-coupled device (CCD), images of the reflected light from the sample points can be obtained, allowing for dynamic or static analysis. As a new high-throughput microbiological analysis method, surface plasmon resonance imaging can simultaneously and dynamically monitor every instantaneous change during biomolecular interactions, thereby revealing the mechanism of biomolecular action. Compared to other bioanalytical techniques, SPRi offers the unique advantage of label-free or non-destructive detection. SPRi technology is also well-suited for the screening, diagnosis, and detection of genetic diseases.

[0085] In this disclosure, "sample" and "specimen" can be used interchangeably. "Sample" or "specimen" can be serum or plasma, or any sample obtained from a patient, including but not limited to: whole blood, urine, saliva, cerebrospinal fluid and platelets.

[0086] In this disclosure, "affinity" and "binding affinity" are used interchangeably. "Affinity" and "binding affinity" refer to the tendency or strength of a polypeptide probe to bind to an analyte. For example, "affinity" can have an equilibrium dissociation constant (K D ), dissociation rate (K d ) or binding rate (K a )express.

[0087] In this disclosure, "magnetic beads" refers to beads or particles that can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuONiO / Fe. Examples of ferrimagnetic materials include NiFe2O4CoFe2O4Fe3O4 (or FeO·Fe2O3). The beads can have a solid core portion that is magnetic and surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer around the non-magnetic core.

[0088] The present invention establishes a high-throughput probe library through a hybrid splitting method, and uses magnetic bead primary screening and surface plasmon imaging fine screening to perform high-throughput screening on the probe library to obtain high-affinity, high-specificity probes. The main interaction forces involved in intermolecular interactions are combined to further analyze and optimize the probe structure. The library is coarsely screened through the kinetic curve of molecular interactions to obtain high-affinity candidate polypeptide probes. Combined with the target protein structure, the common characteristics of the candidate probe structure are captured by intermolecular forces such as hydrogen bonds and hydrophobic interactions. By combining the epitope sequence, the intermolecular forces related to the corresponding probe binding domain are amplified to optimize the probe structure. The specific optimization process and ideas are detailed in the examples. Then, microfluidic chip technology is combined to construct a glass-based microarray diagnostic chip and a diagnostic kit to achieve early diagnosis of Huntington's disease.

[0089] The present disclosure provides a polypeptide probe that specifically binds to a mutant huntingtin protein, which has the following structure from N-terminus to C-terminus: M-(AA1)nN-(AA2)m, wherein M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, and m<5.

[0090] The present disclosure further provides a method for preparing the aforementioned polypeptide probe that specifically binds to huntingtin protein, the method comprising the following steps:

[0091] i) constructing a peptide probe library using a hybrid splitting method,

[0092] ii) Using magnetic beads for primary screening, peptide probes were enriched using mutant huntingtin protein.

[0093] iii) optimizing the polypeptide probe, comprising:

[0094] a) The polypeptide probe has the following structure from N-terminus to C-terminus:

[0095] M-(AA1) n -N-(AA2) m ,

[0096] Wherein, M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, m<5,

[0097] b) Using molecular dynamics curves, the library is coarsely screened to obtain high-affinity candidate peptide probes.

[0098] c) Combined with the target protein structure, the common structural characteristics of candidate probes are captured by intermolecular forces such as hydrogen bonds and hydrophobic interactions. By combining the epitope sequence, the intermolecular forces related to the corresponding probe binding domain are amplified to further optimize the peptide probe structure.

[0099] The following Example 1 provides the specific process and steps for the synthesis, screening and optimization of a polypeptide probe library that specifically binds to mutant huntingtin protein (mHtt).

[0100] Example 1. Synthesis, screening and optimization of a peptide probe library specifically binding to mutant huntingtin protein (mHtt)

[0101] 1. Synthesis and initial screening of peptide probe library

[0102] The hybrid split method was used to construct a peptide probe library. The specific steps are as follows:

[0103] 1) Weigh 150 mg of Tentagel-NH2 resin and follow the solid-phase peptide synthesis protocol (immerse the resin in 10% dihydropyridine (DMF) solution for deprotection and sufficient swelling; rinse the resin three times with DMF before proceeding to the next reaction; add the amino acid to the resin in a 1:1 mixture of HBTU and the mixture to the reaction tube; after completion, add dihydropyridine for deprotection and proceed to the next cycle). Divide the resin into four equal portions, add 180 mg of Asn, Gln, Ser, and Lys to each tube, and react for three cycles in sequence.

[0104] 2) After the reaction is complete, the resin is divided into four equal parts. 60 mg of Asn, Gln, Thr, Arg and an equal amount of HBTU are added to each tube for coupling. After the coupling is complete, the four tubes of resin are mixed and deprotected.

[0105] 3) Divide the resin into four equal parts, add 60 mg of Trp, Leu, Phe, His and an equal amount of HBTU to each tube for coupling. After coupling is complete, mix the four tubes of resin and deprotect;

[0106] 4) The resin was then divided into four equal parts. 45 mg of Arg, Gln, Trp, Phe and an equal amount of HBTU were added to each tube for coupling. After the coupling was completed, the four tubes of resin were mixed and deprotected.

[0107] 5) Divide the resin into four equal parts and add 36 mg of Gln, Gly, Asn, Ser and an equal amount of HBTU to each tube for coupling. After coupling is complete, mix the four tubes of resin and deprotect.

[0108] 6) The resin was then divided into four equal parts. 36 mg of Phe, Ser, Gln, and Tyr were added to each tube along with an equal amount of HBTU for coupling. After the coupling was complete, the four tubes of resin were mixed and deprotected.

[0109] 7) After the above steps, methanol is used for solvent replacement and resin shrinkage, and the resin is vacuum-dried to obtain a dry resin loaded with the peptide library;

[0110] 8) Then, the peptide chain is cleaved from the resin under strong acid and the side chain protecting groups are removed to obtain the probe;

[0111] 9) mHtt was linked to magnetic beads modified with carboxyl groups at the end using EDCNHS in a 1:1 ratio. After thorough washing with PBS, the modified magnetic beads were mixed with the probe library and allowed to interact for 30 minutes. The bound probes were enriched using a magnetic field to obtain affinity binding probes for preliminary screening.

[0112] The design of the mHtt probe library is based on the following considerations: the target protein mHtt is a secreted protein with a certain degree of hydrophilicity. Therefore, the design of the probe library needs to consider the input of hydrophilic residues, while considering intermolecular hydrogen bonds and electrostatic interactions. Therefore, the first two rounds of input correspond to Asn, Gln, Ser, Lys, Asn, Gln, Thr, and Arg. However, mHtt also has hydrophobic residues, and these residues play an important role in its β-sheet structure and appear interspersed. Therefore, hydrophobic interactions and π-π stacking need to be considered in subsequent inputs. Therefore, each subsequent round of amino acid unit input needs to cover the above-mentioned hydrophilic and hydrophobic residues. At the same time, considering the steric hindrance of probe binding, it is necessary to avoid large-scale repetition of benzene rings in the side chains, large-scale repetition of functional groups with the same charge, and avoid thiol side chains with an interval of more than 3 residues. Therefore, residues such as Phe should be avoided as much as possible to avoid intramolecular folding interfering with binding.

[0113] According to the intermolecular interactions, the residues of several types of intermolecular interactions (hydrogen bonds, electrostatic forces, hydrophobic interactions, π-π stacking) that may be involved are included. According to the excess reaction and molecular weight, the above preparation scheme is formulated by comprehensively considering factors such as reaction efficiency and grafting difficulty caused by molecular weight and steric hindrance.

[0114] The principle diagram of the initial screening of probes is shown in Figure 1. Next, surface plasmon resonance imaging technology was used to further screen the candidate probes.

[0115] 2. Probe Screening

[0116] High-throughput screening of the synthesized probes was performed using surface plasmon resonance imaging technology. The specific steps are as follows:

[0117] 1) Dissolve the synthetic probe library in ddH2O to a concentration of 100 μg / mL;

[0118] 2) Spot the sample onto a bare gold chip, repeating the process three times for each sample. Incubate at 4°C for 12 hours, then block the chip with 5% skim milk at 4°C for 12 hours. Rinse thoroughly with 10X PBS, 1X PBS ultrapure water, and blow dry with nitrogen.

[0119] 3) Install the chip on the SPRi instrument, measure the SPRi angle and adjust it to the optimal optical position. Select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL / s.

[0120] 4) PBS was selected as the buffer solution and passed through the flow cell until the baseline stabilized. Then, mHtt was detected in sequence at concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, with an association time of 300 seconds and a dissociation time of 300 seconds. Phosphoric acid was passed between each concentration for regeneration.

[0121] The test results are shown in Figure 2. In Figure 2, from top to bottom, the test results for probes coded mHBP265, mHBP1087, and mHBP3296 are shown. In each test result graph, the curves from bottom to top represent the test results for mHtt concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The amino acid sequence of the probe obtained through high-throughput screening is:

[0122] mHBP265: (Asn-Gln-Phe-Gln-Gln-Gln);

[0123] mHBP1087: (Gln-Gln-His-Gln-Gln-Gln); and

[0124] mHBP3296: (Lys-Gln-Phe-Gln-Gln-Gln).

[0125] After fitting, the equilibrium dissociation constants K D 5.32×10 -7 mol / L、6.88×10 -7 mol / L and 9.89×10 -7 mol / L. It can be seen that the significant feature of the mHtt probe is that the C-terminus selectively binds to the glutamine (Gln) repeat region (i.e., polyQ).

[0126] 3. Probe Optimization

[0127] The commonality of the probe structure was captured, namely AA1-Gln-AA2-Gln3. The most significant feature, -Gln3, was retained in further optimization. The N-terminal feature AA1-Gln-AA2 is a typical dual-coupled binding domain feature. However, the rough screening results did not reveal the specific optimal AA1 and AA2. At the same time, considering that the protein binding epitope is large, -Gln- may not be the optimal length. Without introducing complex side chain groups and constructing super secondary structures, the length of -Gln- was adjusted, and AA1 and AA2 types were simultaneously screened. Therefore, the C-terminus maintained the optimal solution Gln-Gln-Gln, and the N-terminal hydrophilic chain was further extended. The probe was designed as follows, and the synthesis method was carried out according to the above-mentioned peptide synthesis method:

[0128] mHBPL1: Asn-Gln5-Phe-Gln-Gln-Gln;

[0129] mHBPL2: Gln-Gln5-His-Gln-Gln-Gln;

[0130] mHBPL3: Lys-Gln5-Phe-Gln-Gln-Gln;

[0131] mHBPL4:Lys-Gln 10 -Phe-Gln-Gln-Gln;

[0132] mHBPL5:Gln-Gln 10 -His-Gln-Gln-Gln; and

[0133] mHBPL6:Asn-Gln 10 -Phe-Gln-Gln-Gln.

[0134] The performance of the synthesized probe was characterized using surface plasmon resonance imaging technology. The specific steps are as follows:

[0135] 1) Dissolve the synthetic probe library in ddH2O to a concentration of 100 μg / mL;

[0136] 2) Spot the sample onto a bare gold chip (replicate three times for each sample). Incubate at 4°C for 12 hours. Block the chip with 5% skim milk at 4°C for 12 hours. Rinse thoroughly with 10X PBS, 1X PBS ultrapure water, and blow dry with nitrogen.

[0137] 3) Install the chip on the SPRi instrument, measure the SPRi angle and adjust it to the optimal optical position. Select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL / s.

[0138] 4) PBS was selected as the buffer solution and passed through the flow cell until the baseline stabilized. Then, mHtt was detected in sequence at concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, with an association time of 300 seconds and a dissociation time of 300 seconds. Phosphoric acid was passed between each concentration for regeneration.

[0139] The test results are shown in Figure 3. Figure 3 shows the test results for probes mHBPL1, mHBPL2, mHBPL3, mHBPL4, mHBPL5, and mHBPL6. In each test result graph, the curves from bottom to top represent the test results for mHtt concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. After fitting, the equilibrium dissociation constants KD of mHBPL1-6 were 0.96 × 10 -7 mol / L、1.52×10 -9 mol / L、4.65×10 -9 mol / L、8.28×10 -8 mol / L、6.59×10 -8 mol / L、4.32×10 -10 mol / L, it can be seen that mHBPL6 is stronger than mHBPL1, and both are stronger than the mHBP series probes in the above screening steps, thus proving the correctness of the optimization strategy. The notable feature of the mHtt probe is that the N-terminal fragment contains Asn-polyQ.

[0140] Because the interaction epitope between peptide probes and target proteins is very broad, if the interaction is at the appropriate site, it will not be limited to 1-3 residues, but can be further amplified. Screening the library found that probes with hydrophilic C-termini all have strong affinity. Therefore, combined with the above principles, there is further sufficient reason to amplify this interaction by increasing the number of corresponding residues and further enhance the effect. However, it is not clear what number of Gln is more appropriate. Generally speaking, peptide probes are generally constrained to within 20 residues, because too many amino acid residues will also lead to overly complex secondary structures or even super-secondary structures, thereby limiting interactions or making regulation difficult due to factors such as steric hindrance. In addition, probes that are too long are difficult and costly to synthesize, which does not meet the requirements of probe design. Therefore, two nodes of 5 and 10 are used to regulate within a reasonable length range. The stronger the affinity, the higher the capture efficiency.

[0141] If the C-terminus is amplified, it simply extends the hydrophilic Gln tail, and its action site is different. Unless the target protein also has corresponding residues with ultra-high repetitiveness, there will be no clear effect. However, due to its structural stability, natural secretory proteins rarely have ultra-long repetitive fragments. Therefore, this plan adopts N-terminal extension. However, the main purpose of N-terminal optimization here is not to extend the hydrophilic binding length, because the size of the binding epitope is not infinite. Therefore, the biggest purpose of this plan is to enhance hydrogen bonds while placing Asn and Phe at the optimal spatial binding distance to enhance affinity.

[0142] Because Gln residues have no side chain functional groups, the binding mainly comes from the interaction between the side chain functional groups and the target, and it is not possible to introduce new binding interactions while enhancing hydrophilicity. Therefore, repeated Gln residues are selected instead of other hydrophilic residues.

[0143] The present disclosure also provides a bivalent or multivalent assembly of a polypeptide probe that specifically binds to a mutant huntingtin protein (mHtt), wherein the bivalent or multivalent assembly is formed by covalently linking via a linker molecule, or by mixing with a multimer and non-covalently linking. Specifically, the polypeptide probe that specifically binds to a mutant huntingtin protein (mHtt) provided in an embodiment of the present disclosure can be dissolved in ddH2O with the aid of a solvent and incubated at a constant temperature to obtain an assembly.

[0144] The present disclosure provides a polypeptide probe that specifically binds to a neurofilament light chain, which has the following structure from the N-terminus to the C-terminus: XY-(Ala) n -Z-Thr-Ser or XY-(Gly) n -Z-Thr-Ser, wherein the amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, and n is less than 10. The present disclosure also provides a method for preparing the above-mentioned polypeptide probe that specifically binds to neurofilament light chain, the method comprising the following steps:

[0145] i) constructing a peptide probe library using a hybrid splitting method,

[0146] ii) Using magnetic beads for primary screening, the neurofilament light chain was used to enrich the peptide probes.

[0147] iii) optimizing the polypeptide probe, comprising:

[0148] a) The polypeptide probe has the following structure from N-terminus to C-terminus:

[0149] XY-(Ala) n -Z-Thr-Ser or XY-(Gly) n -Z-Thr-Ser,

[0150] The amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, n < 10,

[0151] b) Using molecular dynamics curves, the library is coarsely screened to obtain high-affinity candidate peptide probes.

[0152] c) Considering steric hindrance and intermolecular forces, the distance between the hydrophilic end and the hydrophobic end is increased to further extend the N-terminal hydrophobic chain.

[0153] The following Example 2 provides the specific process and steps for the synthesis, screening and optimization of a polypeptide probe library that specifically binds to neurofilament light chain (NfL).

[0154] Example 2. Synthesis, screening and optimization of a peptide probe library specifically binding to neurofilament light chain (NfL)

[0155] 1. Synthesis and initial screening of peptide probe library

[0156] The hybrid split method was used to construct a peptide probe library. The specific steps are as follows:

[0157] 1) Weigh 150 mg of Tentagel-NH2 resin and follow the solid-phase peptide synthesis cycle (immerse the resin in 10% dihydropyridine (DMF) solution for deprotection and sufficient swelling; rinse the resin three times with DMF before proceeding to the next reaction; add the amino acid to the reaction tube containing the resin in a 1:1 ratio of HBTU and react; after completion, add dihydropyridine for deprotection and proceed to the next cycle). Then, add 180 mg of Met, Tyr, and Leu in sequence and react for three cycles.

[0158] 2) After the reaction is complete, the resin is divided into three equal parts. 60 mg of Trp, Gly, and Tyr and an equal amount of HBTU are added to each tube for coupling. After the coupling is complete, the three tubes of resin are mixed and deprotected.

[0159] 3) Divide the resin into three equal parts, add 60 mg of Ala, Cys, His and an equal amount of HBTU to each tube for coupling. After coupling is complete, mix the three tubes of resin and deprotect;

[0160] 4) The resin was then divided into three equal parts. 45 mg of Ile, Asn, and Gln and an equal amount of HBTU were added to each tube for coupling. After the coupling was completed, the three tubes of resin were mixed and deprotected.

[0161] 5) Divide the resin into three equal parts, add 36 mg of Lys, Thr, and Met and an equal amount of HBTU to each tube for coupling. After coupling is complete, mix the three tubes of resin and deprotect;

[0162] 6) Divide the resin into three equal parts, add 36 mg of Val, Ser, and Met and an equal amount of HBTU to each tube for coupling. After coupling is complete, mix the three tubes of resin and deprotect;

[0163] 7) After the above steps, methanol is used for solvent replacement and resin shrinkage, and the resin is vacuum-dried to obtain a dry resin loaded with the peptide library;

[0164] 8) Then, the peptide chain is cleaved from the resin under strong acid and the side chain protecting groups are removed to obtain the probe;

[0165] 9) NfL was linked to magnetic beads modified with carboxyl groups at the end using EDC:NHS 1:1. After thorough washing with PBS, the modified magnetic beads were mixed with the probe library and allowed to interact for 30 minutes. The bound probes were enriched using a magnetic field to obtain affinity binding probes for preliminary screening.

[0166] The design of the NfL probe library is based on the following considerations: In combination with the NfL structure, the probe design concept is amphiphilic polypeptide, so the ratio of hydrophilic to lipophilic amino acids needs to be controlled at each step of amino acid addition. However, at the same time, the high density of benzene rings and alkane side chains in the NfL protein is taken into consideration, so residues such as Tyr and Leu that can produce hydrophobic interactions or π-π stacking with it are focused on.

[0167] 2. Probe Screening

[0168] High-throughput screening of the synthesized probes was performed using surface plasmon resonance imaging technology. The specific steps are as follows:

[0169] 1) Dissolve the synthetic probe library in ddH2O to a concentration of 100 μg / mL;

[0170] 2) Spot the sample onto a bare gold chip (replicate three times for each sample). Incubate at 4°C for 12 hours. Block the chip with 5% skim milk at 4°C for 12 hours. Rinse thoroughly with 10X PBS, 1X PBS ultrapure water, and blow dry with nitrogen.

[0171] 3) Install the chip on the SPRi instrument, measure the SPRi angle and adjust it to the optimal optical position. Select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL / s.

[0172] 4) PBS was selected as the buffer solution and passed through the flow cell until the baseline stabilized. Then, NfL at concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM was detected in sequence with an association time of 300 seconds and a dissociation time of 300 seconds. Phosphoric acid was passed between each concentration for regeneration.

[0173] The test results are shown in Figure 4. In Figure 4, from top to bottom, the test results for probes NBP596, NBP775, and NBP4061 are shown. In each test result graph, the curves from bottom to top represent the test results for NfL concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The amino acid sequence of the probe obtained through high-throughput screening is:

[0174] NBP596: Met-Trp-Ala-Gln-Thr-Ser;

[0175] NBP775: Met-Trp-Ala-Asn-Thr-Ser; and

[0176] NBP4061: Leu-Tyr-Ala-Asn-Thr-Ser.

[0177] After fitting, the equilibrium dissociation constants KD of the three peptide probes were 3.31×10 -7 mol / L, 2.06×10 -7 mol / L、1.71×10 -7 mol / L. It can be seen that the significant feature of the NfL probe is that the N-terminus is hydrophobic and the C-terminus has hydrophilic residues -Thr-Ser.

[0178] 3. Probe Optimization

[0179] Considering the steric hindrance and intermolecular forces, a certain molecular distance is required between hydrophilic and hydrophobic molecules. Therefore, the probe structure was optimized according to this model, with the N-terminus maintaining the optimal Leu-Tyr-Ala scheme and the N-terminal hydrophobic chain further extended. The probe was designed as follows, and the synthesis method was carried out according to the above-mentioned peptide synthesis method:

[0180] NBPL1: Leu-Tyr-Ala3-Asn-Thr-Ser;

[0181] NBPL2: Leu-Tyr-Ala5-Asn-Thr-Ser;

[0182] NBPL3:Leu-Tyr-Ala7-Asn-Thr-Ser; and

[0183] NBPL4:Leu-Tyr-Ala9-Asn-Thr-Ser.

[0184] The performance of the synthesized probe was characterized using surface plasmon resonance imaging technology. The specific steps are as follows:

[0185] 1) Dissolve the synthetic probe library in ddH2O to a concentration of 100 μg / mL;

[0186] 2) Spot the sample onto a bare gold chip (replicate three times for each sample). Incubate at 4°C for 12 hours. Block the chip with 5% skim milk at 4°C for 12 hours. Rinse thoroughly with 10X PBS, 1X PBS ultrapure water, and blow dry with nitrogen.

[0187] 3) Install the chip on the SPRi instrument, measure the SPRi angle and adjust it to the optimal optical position. Select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL / s.

[0188] 4) PBS was selected as the buffer solution and passed through the flow cell until the baseline stabilized. Then, NfL at concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM was detected in sequence with an association time of 300 seconds and a dissociation time of 300 seconds. Phosphoric acid was passed between each concentration for regeneration.

[0189] The test results are shown in Figure 5. Figure 5 shows the test results for probes NBPL1, NBPL2, NBPL3, and NBPL4. In each test result graph, the curves from bottom to top represent the test results for NfL concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The equilibrium dissociation constants KD for NBPL1-4 were fitted to be 9.54 × 10 -8 mol / L、7.68×10 -9 mol / L、4.21×10 -10 mol / L、6.22×10 -8 mol / L, NBPL3 is stronger than NBPL4, and both are stronger than the NBP series probes in the above screening step 2, which proves the correctness of the optimization strategy. Affected by steric hindrance and intermolecular forces, hydrophilic and hydrophobic molecules need to have a certain molecular distance.

[0190] The present disclosure also provides a bivalent or multivalent assembly of a polypeptide probe that specifically binds to neurofilament light chain (NfL), wherein the bivalent or multivalent assembly is formed by covalently linking a linker molecule, or by mixing with a polymer and non-covalently linking. Specifically, the polypeptide probe that specifically binds to mutant huntingtin protein (mHtt) provided in the embodiments of the present disclosure can be dissolved in ddH2O with the aid of a solvent and incubated at a constant temperature to obtain an assembly.

[0191] Example 3. Kit preparation

[0192] The present disclosure provides a kit comprising at least one of the aforementioned polypeptide probes that specifically bind to mutant huntingtin protein and polypeptide probes that specifically bind to neurofilament light chain.

[0193] The reagent components of the kit include surfactant (Tween 20, sodium dodecyl sulfate SDS, fatty acid sorbitan Span), PBS, EDTA, and Rhodamine B-Probe.

[0194] The surfactant is one or a combination of Tween 20, sodium dodecyl sulfate (SDS) and fatty acid sorbitan Span, which is used to prevent cell aggregation during binding and capture, while improving the binding efficiency of the probe and cell surface antigens. The volume percentage is 0.1%-5%, preferably 0.5%.

[0195] The role of ethylenediaminetetraacetic acid (EDTA) is to maintain stability and resist enzymatic hydrolysis, and the molar concentration is 0.5mmol / L to 10mmol / L.

[0196] The Rhodamine B-labeled polypeptide probe (Rhodamine B-Probe) is the functional main body, and the molar concentration is 0.5 mmol / L to 50 mmol / L.

[0197] PBS is the buffer system of the entire kit, with a pH value of 7.4.

[0198] Optionally, the volume ratio of sodium dodecyl sulfate (SDS) and fatty acid sorbitan Span is 1:1.5, that is, 0.5% SDS + 0.75% Span. Because the probe has a certain degree of hydrophobicity, it is easy to settle during testing or later use. After sedimentation, the reaction efficiency between the solid phase and the liquid phase is low and cannot effectively react (industry consensus), so it is necessary to maintain its stable liquid phase system. When the volume ratio of sodium dodecyl sulfate (SDS) and fatty acid sorbitan Span is 1:1.5, the probe can be effectively dispersed and prevented from settling.

[0199] The various components of the test kit are optionally provided in suitable containers when necessary. The test kit may also include a container for holding or storing a sample (e.g., a container or cartridge for urine, saliva, plasma, cerebrospinal fluid, or serum samples, or a suitable container for storing, transporting, or processing tissue to create a tissue aspirate). Where appropriate, the test kit may also optionally contain a reaction vessel, a mixing vessel, and other components that facilitate the preparation of reagents or test samples. The test kit may also include one or more sample collection / acquisition instruments for assisting in obtaining a test sample, such as various blood collection / transfer devices such as microsampling devices, microneedles, or other minimally invasive painless blood collection methods; blood collection tubes; lancets; capillary blood collection tubes; other blood collection methods that pierce a single fingertip; oral swabs, nose / throat swabs, etc., annular blades for punch biopsy (e.g., 1-8 mm or other suitable sizes), surgical scalpels or lasers (e.g., particularly handheld), syringes, sterile containers, or cannulas for obtaining, storing, or aspirating tissue samples, etc. The kit may include one or more instruments for assisting in joint aspiration, cone biopsy, punch biopsy, fine needle aspiration biopsy, image-guided percutaneous needle biopsy, bronchoalveolar lavage, endoscopic biopsy, and laparoscopic biopsy.

[0200] Example 4. Preparation of probe array microfluidic chip

[0201] Microfluidics is a technology that precisely controls and manipulates fluids at the microscale. This technology enables the integration of essential analytical processes—sample preparation, reaction, separation, and detection—on a centimeter-scale chip. Microfluidics is commonly used in the analysis of trace quantities of pharmaceuticals in biology, chemistry, and medicine. Microfluidic devices offer significant advantages, including low sample consumption, rapid detection, ease of operation, multifunctional integration, compact size, and portability. They hold enormous potential for application in these fields.

[0202] "Microfluidic chip" is a general term for a reaction platform for performing chemical, biochemical, proteomic or molecular tests, which may also be required in medical diagnosis, drug testing, etc. Typically, a microfluidic chip includes an inert matrix such as silicon, glass or ceramic (usually about 1 cm or less in surface area) on which one or more reaction sites are provided. The sites usually carry one or more ligands, such as one or more antibodies or probes, which are selected for the test (or analysis) to be implemented and are activated by adsorption to the chip surface by binding to the sample (such as a blood sample) and / or reagent applied to the microfluidic chip. Many optional techniques can be used to detect the reaction, including chemiluminescence produced by the detection reaction. Some microfluidic chips carry a large number (hundreds or thousands) of such test sites, which are usually arranged in a grid or array to make it possible to perform multiple analyses simultaneously using the same sample.

[0203] The motion of fluid droplet in microfluidic chip can be realized by any suitable means.For example, using fluid manipulation force, for example, electrowetting, dielectrophoresis, light-induced electrowetting, electrode mediation, electric field mediation, electrostatic drive etc. or their combination, fluid droplet can be made to move in microfluidic device or module.Fluid droplet is connected (for example, flow channel) from microfluidic module to the motion of nanopore module by fluid and can be via diffusion, Brownian motion, convection, pumping, applied pressure, gravity-driven flow, density gradient, temperature gradient, chemical gradient, pressure gradient (positive or negative), pneumatic pressure, the chemical reaction of producing gas, attentive flow, capillary pressure, wicking, electric field mediation, electrode mediation, electrophoresis, dielectrophoresis, magnetic migration, magnetic field, magnetic force-driven flow, optical force, chemotaxis, phototaxis, the flow driven by surface tension gradient, Marangoni stress, heat-capillary convection, surface energy gradient, acoustic migration, surface acoustic wave, electroosmotic flow, thermal migration, electrowetting, light-induced electrowetting or its combination. Fluid droplets can be moved in the nanopore module and positioned across the nanopore layer via fluid manipulation forces (eg, electrowetting, dielectrophoresis, photoinduced electrowetting, electrode-mediated, electric field-mediated, electrostatic actuation, etc., or combinations thereof).

[0204] As used in this disclosure, "droplet" and "fluid droplet" are used interchangeably to refer to a discrete volume of liquid that is generally spherical in shape and bounded on at least two sides by the walls or base of a microfluidic device, nanopore device, microfluidic module, or nanopore module. "Generally spherical" in the context of a droplet refers to a shape such as a sphere, particularly an oblate spheroid, e.g., a disk, a rod, a truncated sphere, an ellipsoid, a hemisphere, or an oval. The volume of the droplet in the microfluidic and nanopore modules and devices disclosed herein can be in the range of about 10 μL to about 5 pL, e.g., 10 pL-1 pL, 7.5 μL-10 pL, 5 μL-1 nL, 2.5 μL-10 nL, or 1 μL-100 nL, e.g., 10 μL, 1 μL, 800 nL, 400 nL 100 nL, 10 nL, or less.

[0205] The present disclosure provides a method for preparing a functional microfluidic chip coupled with a small molecule probe. A specific embodiment is given below. The microfluidic chip involved is prepared by G2.5 Sputter, PECVD, photolithography and other processes, as follows:

[0206] 1) Microstructure Patterning: The glass was pre-cleaned as standard, followed by spin coating of an OC adhesive (adhesion layer), followed by deposition of a PVX layer (SiO2 1000A + SiNx 2000A). The patterning was then photoprotected and patterned using a coating (30kPa / 300rpm*10s), pre-baking at 90°C for 120s. This was repeated twice, followed by exposure, development for 100s, hard baking at 230°C for 30min, and finally ICP etching.

[0207] 2) The screened probes are coupled to the patterned surface of the reaction area.

[0208] Figure 6 shows a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present disclosure. As shown in Figure 6, the microfluidic chip may include an inlet 100, a flow channel 101, a buffer zone 102, a reaction zone 103, and an outlet 104. The reaction zone of the microfluidic chip may be coupled to at least one of the aforementioned polypeptide probes that specifically bind to mutant huntingtin protein and the polypeptide probes that specifically bind to neurofilament light chain.

[0209] Example 5. Huntington's disease detection

[0210] The probes screened and prepared by the methods in the aforementioned examples were used to detect Huntington's disease using surface plasmon resonance imaging (SPRi) detection methods. The specific steps are as follows:

[0211] 1) Place the chip on the prism and adjust the angle of the incident light to ensure that it is totally reflected at the junction of the chip and the prism;

[0212] 2) Pass whole blood / serum / plasma / cerebrospinal fluid (CSF) samples into the chip at a dilution ratio of 1:10 and a flow rate of 3-10 μL / s.

[0213] 3) Collect the reflected light intensity and use the BIAevaluation software to analyze the changes in reflected light intensity to distinguish normal people from patients.

[0214] The principle of diagnosis is that after the component to be detected in the sample binds to the probe, the surface plasmon resonance causes a change in the refractive index, which leads to a change in light intensity and a difference in the vertical coordinate on the kinetic curve, and then the cutoff value is divided to complete the diagnosis.

[0215] The test results for patients (n=10) and healthy controls (n=10) are shown in Figure 7. In Figure 7, probe 1 represents the peptide probe mHBPL6, which specifically binds to mutant huntingtin protein; probe 2 represents the peptide probe NBPL3, which specifically binds to neurofilament light chain; and the probe combination represents the combination of mHBPL6 and NBPL3. As can be seen, both probe 1 and probe 2 significantly differentiated between the patient and healthy controls. However, at confidence intervals >0.85, their vertical coordinates overlapped at AU 0.7-0.9 and 0.8-0.9, respectively. Therefore, using a single probe as a diagnostic criterion still presents a high risk. Using a dual-probe array significantly improved the discriminatory power, achieving a cutoff value of 0.87, which was sufficient to distinguish patients from healthy controls.

[0216] In addition to the surface plasmon resonance imaging (SPRi) detection method mentioned above, the enzyme-linked immunosorbent assay (ELISA) can also be used. ELISA involves immobilizing a known antigen or antibody on the surface of a solid support, allowing the enzyme-labeled antigen-antibody reaction to proceed on the solid support. This method can utilize the specific binding of the antigen and antibody to qualitatively and quantitatively measure immune reactions. This detection method has the advantages of being rapid, sensitive, simple, and easy to standardize.

[0217] In the present disclosure, "specifically binds to" and "targets to" are used interchangeably. "Specifically binds to" or "binding specificity" means that the binding member binds to the analyte molecule with a specificity sufficient to distinguish between the analyte molecule and other components or contaminants of the test sample. The antibody can be any antibody that is capable of specifically binding to the target analyte. For example, suitable antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, microbodies, domain antibodies (dAbs), and include single domain antibodies (sdAbs), which are naturally occurring, for example, as in cartilaginous fish and camelids, or which are synthetic, for example, nanobodies, VHHs or other domain structures), synthetic antibodies (sometimes referred to as antibody mimics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates") and fragments of each respectively. Binding members can be, for example, antigen-binding fragments (Fab), Fab' fragments, F(ab')2 fragments, recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single domain antibodies, such as variable heavy chain domains ("VHH"; also referred to as "VHH fragments"), recombinant VHH single domain antibodies and VNAR fragments, disulfide-linked Fv ("sdFv") and anti-idiotypic ("anti-Id") antibodies, and functionally active epitope-binding fragments of any of the above, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.

[0218] In some cases, the first binding members can be immobilized on a solid support. "Immobilization" used herein means the stable combination of the first binding members and the surface of a solid support." stable binding " refers to the physical combination between two entities, wherein the average half-life of the combination is one day or more, for example, under physiological conditions. In some aspects, the physical combination between two entities has an average half-life of 2 days or more, 1 week or more, 1 month or more (including 6 months or more, for example, 1 year or more) at 4 DEG C in PBS. According to some embodiments, the stable combination is derived from a covalent bond between the two entities, a non-covalent bond between the two entities (for example, an ionic bond or a metallic bond) or other forms of chemical attraction, such as hydrogen bonding, van der Waals forces, etc.

[0219] The solid support with the surface having immobilized binding reagent above it can be any convenient surface in plane or non-planar conformation, such as the surface of microfluidic chip, the inner surface of compartment, the outer surface of bead (as defined herein) or the inner surface and / or the outer surface of porous beads.For example, the first binding members can be covalently or non-covalently attached to beads, for example, latex, agarose, sepharose, streptavidin, tosyl activated, epoxy resin, polystyrene, amino beads, amine beads, carboxyl beads etc. In certain embodiments, the beads can be particles, for example, microparticles. In certain embodiments, the microparticles can be about 0.1nm to about 10 microns, about 50nm to about 5 microns, about 100nm to about 1 micron, about 0.1nm to about 700nm, about 500nm to about 10 microns, about 500nm to about 5 microns, about 500nm to about 3 microns, about 100nm to 700nm, or about 500nm to 700nm. The particle of the present invention can be about 4-6 micron, about 2-3 micron or about 0.5-1.5 micron.The particle less than about 500nm is sometimes considered as nanoparticle.Thus, the particle optionally can be the nanoparticle of about 0.1nm to about 500nm, about 10nm to about 500nm, about 50nm to about 500nm, about 100nm to about 500nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm or about 500nm.

[0220] In some embodiments, the binding affinity and / or specificity of the first binding members can be increased by changing the binding buffer. In some embodiments, the binding affinity and / or specificity of the first binding members can be increased by changing the binding buffer. In some embodiments, the binding affinity and / or specificity of the first binding members can be increased by changing the binding buffer. In some embodiments, the binding affinity and / or specificity of the first binding members can be increased by changing the binding buffer.

[0221] The binding affinity and / or specificity of the first binding members and / or the second binding members can be measured. In certain embodiments, an aliquot of a sample is measured using one set of conditions, and compared with another aliquot of the sample measured using different groups of conditions, thus determining the impact of the conditions on binding affinity and / or specificity. For example, the conditions may be changed or may be one or more of the following: removing the target analyte from the sample, adding a molecule that competes with the target analyte or ligand, and changing pH, salt concentration or temperature. Additionally or alternatively, the duration may be variable, and changing the conditions may include waiting for the duration before performing the detection method again.

[0222] The solid support may also comprise a protective, blocking, or passivating layer that can eliminate or reduce nonspecific attachment of non-capture components (e.g., analyte molecules, binding members) to the binding surface during the assay, which can result in false positive signals or loss of signal during detection.

[0223] Examples of materials that can be used to form the passivation layer in certain embodiments include, but are not limited to: polymers, such as polyethylene glycol, which exclude nonspecific binding of proteins; naturally occurring proteins with this property, such as serum albumin and casein; surfactants, for example, zwitterionic surfactants such as sulfobetaines; naturally occurring long-chain lipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

[0224] contacting a sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member includes a cleavable tag attached thereto; removing the second binding member that is not bound to the analyte bound to the first binding member; cleaving the tag attached to the second binding member, which binds to the analyte bound to the first binding member; transferring the cleaved tag through or across one or more nanopores in a layer; detecting or measuring the tag transferred through the layer; and evaluating the tag transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tag transferred through the layer detects the analyte present in the sample.

[0225] Elements that can be part of, all of, combined with, or attached to a tag or label include: nanoparticles; gold particles; silver particles; silver, copper, zinc, or other metal coatings or deposits; polymers; drag tags (as defined herein); magnetic particles; floatable particles; metal particles; charged moieties; dielectrophoretic tags, silicon dioxide (e.g., quartz, glass, etc.) with and without impurities; polymethyl methacrylate (PMMA); polyimide; silicon nitride; gold; silver; quantum dots (including CdS quantum dots); Carbon dots; fluorophores; quenchers; polymers; polystyrene; Janus particles; scattering particles; fluorescent particles; phosphorescent particles; spheres; cubes; insulators; conductors; barcoded or labeled particles; porous particles; solid particles; nanoshells; nanorods; microspheres; analytes such as viruses, cells, parasites, and organisms; nucleic acids; proteins; molecular recognition elements; spacers; PEG; dendrimers; charge-altering agents; magnetic materials; enzymes; DNA, including aptamer sequences; amplifiable DNA; repetitive DNA sequences; fusions or conjugates of detectable elements with molecular recognition elements (e.g., engineered binding members); anti-antibody aptamers; aptamers to antibody-binding proteins; absorbed or adsorbed detectable compounds; heme; fluorescein; phosphors; azide or alkyne (e.g., terminal or non-terminal alkyne) or other click chemistry partners.

[0226] The present invention establishes a high-throughput probe library through a hybrid splitting method, and uses magnetic bead primary screening and surface plasmon imaging fine screening to perform high-throughput screening on the probe library to obtain high-affinity, high-specificity probes. The main interaction forces involved in intermolecular interactions are combined to further analyze and optimize the probe structure. The library is coarsely screened through the kinetic curve of molecular interactions to obtain high-affinity candidate polypeptide probes. Combined with the target protein structure, the common characteristics of the candidate probe structure are captured by intermolecular forces such as hydrogen bonds and hydrophobic interactions. By combining the epitope sequence, the intermolecular forces related to the corresponding probe binding domain are amplified to optimize the probe structure. The specific optimization process and ideas are detailed in the examples. Then, microfluidic chip technology is combined to construct a glass-based microarray diagnostic chip and a diagnostic kit to achieve early diagnosis of Huntington's disease.

[0227] Compared with the prior art, the present invention has the following beneficial effects:

[0228] (1) Compared with traditional technologies, the technology provided by the present disclosure can achieve early diagnosis of Huntington's disease with simple operation and high sensitivity;

[0229] (2) The kits, microfluidic chip structures, and probe coupling methods provided by the present disclosure are capable of large-scale mass production and are universally applicable to various diseases;

[0230] (3) The probe design and screening method provided by the present disclosure is faster and more economical than the traditional design method, and has the potential for large-scale promotion, providing new strategies and ideas for diagnosis.

[0231] The applicant states that while the above-described embodiments illustrate the process of the present invention, the present invention is not limited to the above-described process steps, nor does it imply that the present invention must rely on the above-described process steps for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0232] In the accompanying drawings, the thickness of certain areas and layers may be exaggerated for clarity. The same reference numerals in the figures represent the same or similar structures, and their detailed descriptions will be omitted. The described features, structures or characteristics may be combined in one or more embodiments in any suitable manner. In the description of the present disclosure, many specific details are provided so as to provide a full understanding of the embodiments of the present disclosure. However, those skilled in the art will appreciate that the technical solutions of the present disclosure can be practiced without one or more of the specific details, or other methods, components, materials, etc. can be adopted. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid obscuring the main technical ideas of the present disclosure.

[0233] It will be understood that although the terms first, second, third, etc. may be used to describe various elements, components, areas, layers and / or parts in this article, these elements, components, areas, layers and / or parts should not be limited by these terms. These terms are only used to distinguish one element, component, area, layer or part from another element, component, area, layer or part. Therefore, the first element, component, area, layer or part discussed above can be referred to as the second element, component, area, layer or part without departing from the teachings of the present disclosure.

[0234] Spatially relative terms such as "row," "column," "under," "above," "left," "right," and the like may be used herein to describe the relationship of one element or feature to another element or feature as illustrated in the figures for ease of description. It will be understood that these spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figure is flipped, the element described as "under other elements or features" will be oriented as "above other elements or features." Thus, the exemplary term "under" can encompass both orientations of above and below. The device can be oriented otherwise (rotated 90 degrees or in other orientations) and the spatially relative descriptors used herein are interpreted accordingly. In addition, it will also be understood that when a layer is referred to as "between two layers," it can be the only layer between the two layers, or one or more intermediate layers may also be present.

[0235] The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. As used herein, the singular forms "one", "an" and "the" are intended to also include the plural forms, unless the context clearly indicates otherwise. It will be further understood that the terms "include" and / or "comprise" when used in this specification specify the presence of the features, wholes, steps, operations, elements and / or parts, but do not exclude the presence of one or more other features, wholes, steps, operations, elements, parts and / or their groups or add one or more other features, wholes, steps, operations, elements, parts and / or their groups. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. In the description of this specification, the description of the reference terms "one embodiment", "another embodiment" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present disclosure. In this specification, the schematic representation of the above terms does not necessarily need to be directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, those skilled in the art may combine different embodiments or examples and features of different embodiments or examples described in this specification without mutual contradiction.

[0236] It will be understood that when an element or layer is referred to as being “on,” “connected to,” “coupled to,” or “adjacent to” another element or layer, it can be directly on, directly connected to, directly coupled to, or directly adjacent to another element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly connected to,” “directly coupled to,” or “directly adjacent to” another element or layer, no intervening elements or layers are present. However, in no case should “on” or “directly on” be interpreted as requiring that one layer completely cover the underlying layer.

[0237] Embodiments of the present disclosure are described herein with reference to schematic illustrations (and intermediate structures) of idealized embodiments of the present disclosure. Because of this, variations in the illustrated shapes, for example as a result of manufacturing techniques and / or tolerances, should be expected. Therefore, embodiments of the present disclosure should not be interpreted as being limited to the specific shapes of the regions illustrated herein, but should include shape deviations, for example, due to manufacturing. Therefore, the regions illustrated in the figures are schematic in nature, and their shapes are not intended to illustrate the actual shape of the regions of the device and are not intended to limit the scope of the present disclosure.

[0238] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the relevant art and / or the context of this specification, and will not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0239] As will be appreciated by those skilled in the art, although the various steps of the method of the present disclosure are described in a particular order in the accompanying drawings, this does not require or imply that the steps must be performed in this particular order unless the context clearly indicates otherwise. Additionally or alternatively, multiple steps can be combined into a single step and / or a single step can be broken down into multiple steps and performed. In addition, other method steps can be inserted between steps. Inserted steps can represent improvements to the method described herein or can be unrelated to the method. In addition, a given step may not be fully completed before the next step begins.

[0240] The above description is merely a specific embodiment of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A polypeptide probe that specifically binds to mutant huntingtin protein, which has the following structure from N-terminus to C-terminus: M-(AA1) n -N-(AA2) m , in, M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, m<5.

2. The polypeptide probe according to claim 1, wherein AA2 is Gln.

3. The polypeptide probe according to claim 2, wherein The polypeptide probe is selected from the following group: mHBP265: (Asn-Gln-Phe-Gln-Gln-Gln); mHBP1087: (Gln-Gln-His-Gln-Gln-Gln); and mHBP3296: (Lys-Gln-Phe-Gln-Gln-Gln).

4. The polypeptide probe according to claim 1 or 2, wherein AA1 is Gln.

5. The polypeptide according to claim 4, wherein The polypeptide probe is selected from the following group: mHBPL1: Asn-Gln5-Phe-Gln-Gln-Gln; mHBPL2: Gln-Gln5-His-Gln-Gln-Gln; mHBPL3: Lys-Gln5-Phe-Gln-Gln-Gln; mHBPL4:Lys-Gln 10 -Phe-Gln-Gln-Gln; mHBPL5: Gln-Gln 10 -His-Gln-Gln-Gln; and mHBPL6:Asn-Gln 10 -Phe-Gln-Gln-Gln。 6. A bivalent or multivalent body formed by assembling the polypeptide probes according to any one of claims 1 to 5, wherein: The divalent or multivalent body is formed by covalently linking a linker molecule, or by mixing with a polymer and non-covalently linking.

7. A polypeptide probe that specifically binds to a neurofilament light chain, which has the following structure from the N-terminus to the C-terminus: XY-(Ala) n -Z-Thr-Ser or XY-(Gly) n -Z-Thr-Ser, in, The amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, and n is less than 10. The polypeptide probe according to claim 7 , wherein XY is Met-Trp or Leu-Tyr.

9. The polypeptide probe according to claim 8, wherein The polypeptide probe is selected from the following group: NBP596: Met-Trp-Ala-Gln-Thr-Ser; NBP775: Met-Trp-Ala-Asn-Thr-Ser; and NBP4061: Leu-Tyr-Ala-Asn-Thr-Ser.

10. The polypeptide probe according to claim 8, wherein The polypeptide probe is selected from the following group: NBPL1: Leu-Tyr-Ala3-Asn-Thr-Ser; NBPL2: Leu-Tyr-Ala5-Asn-Thr-Ser; NBPL3: Leu-Tyr-Ala7-Asn-Thr-Ser; and NBPL4:Leu-Tyr-Ala9-Asn-Thr-Ser.

11. A bivalent or multivalent body formed by assembling the polypeptide probes according to any one of claims 7 to 10, wherein: The divalent or multivalent body is formed by covalently linking a linker molecule, or by mixing with a polymer and non-covalently linking.

12. A kit comprising at least one of the polypeptide probes specifically binding to mutant huntingtin protein according to any one of claims 1 to 5, the bivalent or multivalent formed by assembling the polypeptide probes according to claim 6, the polypeptide probes specifically binding to neurofilament light chain according to any one of claims 7 to 10, and the bivalent or multivalent formed by assembling the polypeptide probes according to claim 11.

13. The kit according to claim 12, wherein The polypeptide probe is labeled with fluorescein or biotin.

14. The kit according to claim 13, wherein The polypeptide probe is labeled with rhodamine B.

15. The kit according to any one of claims 12 to 14, further comprising a surfactant, a buffer and EDTA.

16. The kit according to claim 15, wherein The surfactant is selected from one or a combination of Tween 20, sodium dodecyl sulfate (SDS) and fatty acid sorbitan Span.

17. The kit according to claim 16, wherein The volume ratio of sodium dodecyl sulfate (SDS) and fatty acid sorbitan Span is 1:1.

5.

18. A microfluidic chip, wherein the reaction zone of the microfluidic chip is coupled with at least one of the polypeptide probes specifically binding to mutant huntingtin protein according to any one of claims 1 to 5, the bivalent or multivalent formed by assembling the polypeptide probes according to claim 6, the polypeptide probes specifically binding to neurofilament light chain according to any one of claims 7 to 10, and the bivalent or multivalent formed by assembling the polypeptide probes according to claim 11.

19. A method for preparing a polypeptide probe that specifically binds to Huntington protein according to any one of claims 1 to 5, the method comprising the following steps: i) constructing a peptide probe library using a hybrid splitting method, ii) Using magnetic beads for primary screening, using mutant huntingtin protein to enrich peptide probes, iii) optimizing the polypeptide probe, comprising: a) The polypeptide probe has the following structure from N-terminus to C-terminus: M-(AA1) n -N-(AA2) m , Wherein, M, AA1 and AA2 are all selected from any one of Asn, Gln and Lys, N is selected from any one of Phe and His, n≤10, and m<5. b) Using molecular dynamics curves, the library is roughly screened to obtain high-affinity candidate peptide probes. c) Combined with the target protein structure, the commonalities of the candidate probe structures are captured based on intermolecular forces such as hydrogen bonds and hydrophobic interactions. By combining the epitope sequence, the intermolecular forces related to the corresponding probe binding domain are amplified to further optimize the peptide probe structure.

20. A method for preparing a polypeptide probe that specifically binds to a neurofilament light chain according to any one of claims 7 to 10, the method comprising the following steps: i) constructing a peptide probe library using a hybrid splitting method, ii) Using magnetic beads for primary screening, using neurofilament light chain to enrich peptide probes, iii) optimizing the polypeptide probe, comprising: a) The polypeptide probe has the following structure from N-terminus to C-terminus: X-Y-(Ala) n -Z-Thr-Ser or X-Y-(Gly) n -Z-Thr-Ser, The amino acid residue represented by XY is hydrophobic, Z is Gln or Asn, and n is less than 10. b) Using molecular dynamics curves, the library is roughly screened to obtain high-affinity candidate peptide probes. c) Considering steric hindrance and intermolecular forces, the distance between the hydrophilic end and the hydrophobic end is increased to further extend the N-terminal hydrophobic chain.

21. Use of a polypeptide probe that specifically binds to mutant Huntington protein according to any one of claims 1 to 5 or a bivalent or multivalent assembled from the polypeptide probe according to claim 6 in the preparation of a therapeutic drug, diagnostic reagent, diagnostic kit or developer for Huntington's disease.

22. Use of a polypeptide probe specifically binding to neurofilament light chain according to any one of claims 7 to 10 or a bivalent or multivalent assembled from the polypeptide probe according to claim 11 in the preparation of a drug, a diagnostic reagent, a diagnostic kit or a developer for the prevention or treatment of neurofilament light chain-related diseases.

23. A method for detecting a sample using the microfluidic chip surface plasmon resonance imaging technique according to claim 18, comprising: placing the microfluidic chip on the prism; Adjust the incident light angle so that the incident light is totally reflected at the junction of the chip and the prism; Pass the sample into the chip; Collect reflected light intensity; Software is used to analyze changes in reflected light intensity to distinguish between different samples.

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