CN1375696A - Ultratrace DNA detecting method by means of nano microsphere amplificaltion technology and piezoelectric DNA biosensor - Google Patents

Abstract

A method for detecting ultra-trace DNA by a piezoelectric DNA biosensor with nano-microsphere amplification technology. A thin layer of gold or silver film is coated on a quartz wafer, and then the quartz crystal oscillator is allowed to selectively adsorb DNA. Through DNA molecular hybridization, combined with In order to form double-stranded DNA, nano-microspheres labeled with drugs or biological reagents that can selectively interact with double-stranded DNA are used to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace amounts of target DNA. In the present invention, by marking the nanometer microspheres capable of selectively acting on the double-strand DNA, the formation of the targeted double-strand DNA molecule is confirmed and the mass is amplified, and ultra-trace target DNA is detected. Compared with conventional piezoelectric DNA biosensors, the minimum detection limit can be improved by 2 to 5 orders of magnitude, so it can not only detect ultra-trace target DNA, but also has strong selectivity and high sensitivity.

Description

Method for detecting ultra-trace DNA by piezoelectric DNA biosensor based on nanosphere amplification technology

technical field

The invention relates to a method for detecting ultra-trace DNA by a piezoelectric DNA biosensor with nanometer microsphere amplification technology.

Background technique

Piezoelectric sensors, also known as quartz crystal microbalances (QCM), are based on the piezoelectric effect. The principle of the piezoelectric DNA biosensor is: first coat a thin layer of gold or silver film on the quartz wafer, and then try to make the quartz crystal oscillator selectively adsorb DNA, and hybridize DNA molecules to another DNA containing complementary base sequence. Recognize, combine into double-stranded DNA, and use frequency changes to reflect hybridization. Since QCM can have a frequency response to the increase of nanogram-level mass on the surface, it is a very sensitive mass detection device suitable for detecting the change of single-stranded DNA (ssDNA) before and after hybridization, so it can be applied to DNA biology. sensor research.

In recent years, the application of mass-type quartz crystal microbalance (QCM) as a biosensor has developed rapidly, and it has shown great potential in the identification and determination of nucleic acid molecular hybridization, and has broad application prospects. Although QCM is a sensitive mass detector that can detect sub-nanogram mass changes, in actual detection, the concentration of DNA is far below the range of direct detection by QCM, especially as a biological tool for detecting ultra-trace changes. The sensitivity of the sensor cannot meet the requirements.

Contents of the invention

The present invention aims at the above problems and provides a method for detecting ultra-trace DNA by piezoelectric DNA biosensors with nano-microsphere amplification technology. This method can detect ultra-trace target DNA with high sensitivity.

The technical solution provided by the invention is: a method for detecting ultra-trace DNA by piezoelectric DNA biosensors with nano-microsphere amplification technology. A thin layer of gold or silver film is plated on a quartz wafer, and then the quartz crystal oscillator is selectively adsorbed. DNA is combined into double-stranded DNA (ds-DNA) through hybridization of DNA molecules, and nanospheres labeled with drugs or biological agents that can selectively interact with double-stranded DNA are used to confirm and test the formation of targeted double-stranded DNA molecules. Mass amplification to detect ultra-trace amounts of target DNA.

The above-mentioned nano microspheres are magnetic microspheres or albumin microspheres.

The above-mentioned chemical drugs capable of selecting with DNA double strands are doxorubicin drugs.

The above-mentioned biological reagent capable of selectively acting on DNA double strands is actinomycin D.

The above-mentioned quartz crystal oscillator is immersed in anhydrous methanol solution for 10-60 minutes before use, then ultrasonically cleaned for 1-3 minutes, washed 3-5 times with double distilled water, and dried in the air.

In the present invention, the large-mass nano-microspheres that can selectively act on double-stranded DNA drugs or biological reagents are embedded on the ds-DNA chip electrodes formed after hybridization with the target DNA as amplified markers to form targeted double-stranded DNA molecules. Perform confirmation and mass amplification to detect ultra-trace amounts of target DNA. Compared with conventional piezoelectric DNA biosensors, the minimum detection limit can be improved by 2 to 5 orders of magnitude, so it can not only detect ultra-trace target DNA, but also has strong selectivity and high sensitivity.

Description of drawings

Accompanying drawing is the effect diagram of using the present invention to detect ultra-trace DNA.

Detailed ways

Example 1: Nano-magnetic microspheres modified with actinomycin D were used to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace target DNA.

1. Preparation of actinomycin D-modified nano-magnetic microspheres: Prepare 1.0×10 ^-6 mol/L actinomycin D (ActD) in acetone solution to prepare the cross-linking reaction between amino groups and aldehyde groups. After adding 0.02 g of aldehyde-based magnetic microspheres (commercially available products can be used), the mixture was stirred at 4 °C and reacted for 4 hours. Save for later.

2. Self-assembled α-lipoic acid monolayer film: The quartz wafer was immersed in anhydrous methanol solution for 30 minutes before use, then ultrasonically cleaned for 1 minute, washed 3 times with double distilled water, and dried in the air. Quickly place the cleaned quartz wafer in the Teflon reaction cuvette of QCM, add 0.5 ml of 0.3 mol/L α-lipoic acid ethanol solution, wash with double distilled water after 2 hours, and dry in the air.

Three, the solidification of probe: take out chip electrode from the Teflon reaction cuvette of QCM, drop the hydrochloric acid 1-ethyl-3-(3-dimethylaminopropyl group) carbodiimide of 10 microliters 100mg/mL ( EDC) and 10 microliters of 100mg/Ml N-hydroxysuccinimide (NHS) to the surface of the self-assembled monolayer modified quartz electrode sheet, and placed for 20 minutes. Wash in double distilled water and air dry. Add 10 microliters of ssDNA (5'-XATGGGTATTCAACATTTCCG, X=-NH ₂ ) probe solution (10 ^-5 mol/L) with -NH ₂ at the 5' end on the surface of the wafer gold electrode, and react for 30 minutes. Wash with 1/15 mol/L phosphate buffer solution (PBS, pH=7.0), and air dry for later use.

4. Hybridization and chimerism: put the chip electrode after the above treatment into the Teflon reaction cell of QCM, use 0.2mol/L phosphate buffer solution (pH=7.0, containing 50% formamide) as the hybridization buffer solution, add In the Teflon reaction cell of QCM, after the frequency of the chip is stable (Δf≤±1Hz/min), add 10 microliters of non-complementary target DNA (ncDNA) (5′-GACGTCAGCA), start QCM, record the frequency change as a blank control, About 5 minutes later, 10 microliters of its complementary target DNA (cDNA) (5'-CGGAAATGTTGAATACC) was added. After the frequency change is stable, add 5-20 microliters of Act D or 0.5 mg/mL Act D-modified nanospheres prepared in step 1 to the Teflon reaction cell to make it chimeric with ds-DNA for 15 minutes, and record frequency changes.

Example 2: Using doxorubicin-modified nano-magnetic microspheres to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace target DNA.

Preparation of doxorubicin-modified magnetic microspheres: Prepare an aqueous solution of 2.0×10 ^-6 mol/L doxorubicin (ADM) to prepare for the cross-linking reaction between amino groups and aldehyde groups, and add 0.02 g of aldehyde-based magnetic microspheres Afterwards, the mixture was stirred and reacted at 4°C for 4 hours, the microspheres were washed 3 times with double distilled water on the magnetic stand, 0.5 mL of double distilled water was added, and stored at 4°C for later use. All the other steps are the same as in Example 1.

Its result shows, compared with conventional piezoelectric DNA biosensor, the present invention has greatly improved the sensitivity of QCM (referring to accompanying drawing, among the figure abscissa is time Time/Sec, and ordinate is frequency change Frequency change/Hz, curve 1 is the change of frequency with time after adding ncDNA, curve 2 is the change of frequency with time after adding cDNA, curve 3 is the change of frequency with time after adding ADM, and curve 4 is the change of frequency with time after adding ADM-modified nano-microspheres change), the lowest detection limit dropped to 10 ^-12 ~ 10 ^-13 mol/L.

Example 3: Confirmation and mass amplification of the formation of targeted double-stranded DNA molecules by albumin nanospheres modified with mitoxantrone, and detection of ultra-trace target DNA.

Preparation of mitoxantrone-modified albumin nanospheres: prepare 2.0×10 ^-6 mol/L mitoxantrone aqueous solution to prepare for the cross-linking reaction between amino groups and aldehyde groups, and add 0.02 g of aldehyde-based albumin After microspheres, the mixture was stirred and reacted at 4°C for 4 hours, centrifuged at 12,000 rpm, washed 3 times with double distilled water, added with 0.5 mL of double distilled water, and stored at 4°C for later use. All the other steps are the same as in Example 1.

The present invention can also be labeled with doxorubicin drugs such as doxorubicin, epirubicin and zorubicin, and other anthracycline antineoplastic drugs such as bisantrene, or other drugs or biological reagents that can selectively interact with double-stranded DNA. Nanospheres were used as amplifying markers.

Claims (4)

1. a Nano microsphere amplification piezoelectric DNA biology sensor detects the method for ultratrace DNA, plating skim gold film or silverskin on quartz wafer, allow optionally adsorption of DNA of quartz crystal oscillator then, pass through dna molecule hybridize, be combined into double-stranded DNA, it is characterized in that: will be marked with can with the Nano microsphere of double-stranded DNA selectively acting medicine or biological reagent, the target double chain DNA molecule formed proves conclusively and do quality and amplify, detect the target dna of ultratrace.

2. method according to claim 1 is characterized in that: described Nano microsphere is magnetic microsphere or albumin microsphere.3. method according to claim 1 and 2 is characterized in that: above-mentioned can be adriamycin class medicine with the chemicals of dna double chain selection effect.

4. method according to claim 1 and 2 is characterized in that: above-mentioned can be actinomycin D with the biological reagent of dna double chain selection effect.

5. method according to claim 1 and 2 is characterized in that: quartz crystal oscillator was immersed in absolute methanol solution earlier 10～60 minutes before using, and ultrasonic cleaning is 1～3 minute then, with redistilled water washing 3～5 times, dried again.