CN-121975917-A - Biosensor based on ultraviolet light triggering biological bar code release technology and construction and application thereof

CN121975917ACN 121975917 ACN121975917 ACN 121975917ACN-121975917-A

Abstract

The invention discloses a biosensor based on an ultraviolet light triggering biological bar code release technology (OBCA) and construction and application thereof. The biosensor comprises nucleic acid functionalized silver nanoparticles, nucleic acid functionalized magnetic beads and a CRISPR/Cas12a detection system, wherein DNA bar codes modified on the surfaces of the silver nanoparticles comprise a light cleavable linker, a CRISPR/Cas12a activation sequence and a target capture sequence I, and DNA capture probes comprising a target capture sequence II are immobilized on the surfaces of the magnetic beads through biotin-streptavidin. During detection, the target miRNA mediates two capture sequences to form a sandwich compound, after magnetic separation and thermal denaturation elution, 365nm ultraviolet irradiation releases a DNA bar code fragment, and the Cas12a trans-cleavage fluorescence quenching report probe is activated to generate a signal. According to the method, nucleic acid amplification and reverse transcription are not needed, the detection limits of miR-21, miR-155 and mi-210 are respectively 158.94aM, 763.56aM and 197.82aM, single base mismatch and precursor miRNA can be distinguished, the detection result of a serum sample is highly consistent with that of RT-PCR, and the method is suitable for high-sensitivity quantitative detection of miRNA.

Inventors

FENG SHANGYUAN
YE JIANQING
LIN XUELIANG
FAN MIN
WANG LINGNA
XU LUYUN

Assignees

泉州师范学院

Dates

Publication Date: 20260505
Application Date: 20260204

Claims (10)

1.A biosensor based on ultraviolet light triggering biological bar code release technology is characterized by comprising: (1) The nucleic acid functionalized silver nanoparticle consists of a silver nanoparticle and a DNA bar code anchored on the surface of the silver nanoparticle, wherein the DNA bar code is modified with a photocleavable linker and a sulfhydryl group and comprises a CRISPR/Cas12a activation sequence and a target capturing sequence I; (2) The nucleic acid functionalized magnetic bead consists of a streptavidin modified magnetic bead and a DNA capture probe fixed on the surface of the streptavidin modified magnetic bead, wherein the DNA capture probe is modified with biotin and comprises a target capture sequence II; (3) A CRISPR detection system comprising a Cas12a protein, a crRNA, and a fluorescence quenching reporter probe; The light-cleavable linker is an ultraviolet-sensitive o-nitrobenzyl linker iPCLink, the CRISPR/Cas12a activating sequence is used for specifically combining crRNA to activate trans-cleavage activity of Cas12a protein, the target capturing sequence I is used for specifically hybridizing with one end of target miRNA, the target capturing sequence II is used for specifically hybridizing with the other end of the target miRNA, and the crRNA can specifically identify and combine with the CRISPR/Cas12a activating sequence in the DNA bar code so as to activate nonspecific cleavage effect of the Cas12a protein on a fluorescence quenching report probe.
2. The biosensor of claim 1, wherein in the DNA barcode, the CRISPR/Cas12a activation sequence is linked to the target capture sequence by one or more nucleotide spacer sequences to form a single functional fusion oligonucleotide.
3. The biosensor of claim 1, wherein the crRNA has a sequence of 5'-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3'.
4. The biosensor of claim 1, wherein the fluorescence quenching reporter probe has a sequence of 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'.
5. A method for constructing a biosensor according to any one of claims 1 to 4, comprising the steps of: S1, preparing a silver nanoparticle colloid solution; S2, assembling the activated DNA bar code and silver nano particles, washing and purifying, and then, re-suspending in a binding buffer solution to obtain a nucleic acid functionalized silver nano particle solution; S3, incubating the DNA capture probe and the streptavidin modified magnetic bead, and washing to obtain a nucleic acid functionalized magnetic bead with the surface fixed with the DNA capture probe; S4, mixing and incubating the nucleic acid functionalized magnetic beads, the nucleic acid functionalized silver nanoparticle solution and the target miRNA, so that the target miRNA is hybridized with the target capturing sequence II and the target capturing sequence I respectively through complementary sections at two ends of the target miRNA, and a sandwich compound of the nucleic acid functionalized magnetic beads, the target miRNA and the nucleic acid functionalized silver nanoparticle is formed; s5, performing magnetic separation and washing on the sandwich compound, after purification, dissociating target miRNA through thermal denaturation treatment, releasing nucleic acid functionalized silver nano particles, and then breaking the photo-cleavable linker through ultraviolet irradiation, so as to release a DNA bar code fragment containing the CRISPR/Cas12a activating sequence and the target capturing sequence I from the surface of the silver nano particles; And S6, mixing and incubating the released DNA bar code fragment with a Cas12a-crRNA complex solution, a fluorescence quenching report probe and a reaction buffer solution, wherein a CRISPR/Cas12a activating sequence in the DNA bar code fragment is identified and combined by crRNA, so as to activate trans-cleavage activity of Cas12a protein, and further cleave the fluorescence quenching report probe to generate a fluorescence signal.
6. The method according to claim 5, wherein in the step S2, the binding buffer has a formula of 100mM NaCl,15 mM MgCl 2 , 0.05wt% Tween-20, and pH=7.4 to 7.6.
7. The method according to claim 5, wherein in step S4, the mixed incubation is performed at room temperature for 40 minutes.
8. The method according to claim 5, wherein the mixed incubation is performed at 37℃for 40 minutes in step S6.
9. Use of the biosensor according to any one of claims 1-4, the construction method according to any one of claims 5-8 for miRNA detection for non-disease diagnostic treatment purposes.
10. The use of claim 9, wherein the miRNA includes, but is not limited to, miR-21, miR-155 and miR-210.

Description

Biosensor based on ultraviolet light triggering biological bar code release technology and construction and application thereof Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a biosensor based on an ultraviolet light triggering biological bar code release technology (OBCA) and construction and application thereof. Background MiRNA is an endogenous non-coding RNA with the length of about 18-25 nt, and is combined with a 3' -untranslated region of target gene mRNA to regulate gene expression, so that the miRNA participates in key physiological processes such as cell proliferation, apoptosis, differentiation and the like. Research shows that miRNA has obvious abnormal expression in malignant tumors such as lung cancer, breast cancer and the like (for example, miR-21 is up-regulated by 2-5 times in serum of a lung cancer patient), and is an ideal biomarker for early diagnosis and prognosis evaluation of cancer. The traditional detection method such as RT-qPCR needs to convert miRNA into cDNA through reverse transcription, relies on large-scale equipment such as a fluorescent quantitative PCR instrument, takes time (3-4 hours) to operate, is easily affected by RNA degradation, has detection limit of at most nM level (10 -9 mol/L), is difficult to meet the detection requirement of low-abundance miRNA of early cancer patients, needs a high-concentration sample (mug level total RNA) for Northern blot, has complicated electrophoresis and hybridization steps (24 hours), cannot realize multiple detection, and has limited clinical applicability. Although CRISPR/Cas12a technology is used for nucleic acid detection by virtue of high-specificity trans-cleavage activity, the CRISPR/Cas12a technology still needs to be reversely transcribed into cDNA and amplified by methods such as RPA, LAMP and the like in miRNA detection, exogenous enzymes and complex primer designs are introduced, the operation steps (> 2 hours) and the risk of false positives are increased, and the direct detection sensitivity is only pM grade (10 -12 mol/L). Although the nano biological bar code technology improves the sensitivity through a signal amplification strategy, the DNA double-chain denaturation is incomplete due to heat release, the chemical release (such as DTT) relies on thiol reagent to compete for replacement, the time is consumed, the downstream reaction is interfered, the existing light-operated release scheme lacks space-time precise regulation, the ultraviolet light utilization rate is low, and the nano particles have obvious interference on the light signal absorption. Therefore, development of a highly sensitive and rapid miRNA detection technique without reverse transcription and additional amplification is an urgent need in the clinical diagnosis field. Disclosure of Invention The invention aims to provide a biosensor based on an ultraviolet light triggering biological bar code release technology (OBCA), and construction and application thereof. The technical scheme adopted by the invention is as follows: a biosensor based on ultraviolet light triggered bio-barcode release technology, comprising: (1) The nucleic acid functionalized silver nanoparticle consists of a silver nanoparticle and a DNA bar code anchored on the surface of the silver nanoparticle, wherein the DNA bar code is modified with a photocleavable linker and a sulfhydryl group and comprises a CRISPR/Cas12a activation sequence and a target capturing sequence I; (2) The nucleic acid functionalized magnetic bead consists of a streptavidin modified magnetic bead and a DNA capture probe fixed on the surface of the streptavidin modified magnetic bead, wherein the DNA capture probe is modified with biotin and comprises a target capture sequence II; (3) A CRISPR detection system comprising a Cas12a protein, a crRNA, and a fluorescence quenching reporter probe; The light cleavable linker is an ultraviolet sensitive o-nitrobenzyl linker iPCLink, the CRISPR/Cas12a activating sequence is used for specifically combining crRNA to activate trans-cleavage activity of Cas12a protein, the target capturing sequence I is used for specifically hybridizing with one end of target miRNA, the target capturing sequence II is used for specifically hybridizing with the other end of the target miRNA, the crRNA can specifically identify and combine with the CRISPR/Cas12a activating sequence in the DNA bar code, and then activate nonspecific cleavage effect of Cas12a protein on a fluorescence quenching report probe; In the DNA barcode, the CRISPR/Cas12a activating sequence is linked to the target capture sequence by one or more nucleotide spacer sequences to form a single functional fusion oligonucleotide; the crRNA has a sequence of 5'-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3'. The sequence of the fluorescence quenching report probe is 5'-FAM-TTTTTTTTTTTTTTT-BHQ1-3'. The method for constructing the biosensor comprises the foll