Construction Of Optical Biosensors Based On DNA Probes And Its Application

As a commonly used analysis and detection method,the molecular spectroscopy analysis method can qualitatively and quantitatively analyze complex samples,has the advantages of simple operation and real-time detection,and has application potential.Among them,fluorescence spectroscopy,ultraviolet-visible absorption spectroscopy,and scattering spectroscopy are widely used in environmental monitoring,disease treatment and other fields.DNA has good stability,programmability,and can be designed into different structures through the principle of base complementary pairing.What’s more,it can be used as a molecular probe to specifically identify the target and is often combined with nucleic acid signal amplification technology to achieve sensitive detection of the target.In this paper,DNA is used as a molecular recognition probe,combined with nanomaterials or nucleic acid signal amplification technology to construct the colorimetric and fluorescent single-signal optical biosensors and the fluorescence and scattering dual-signal optical biosensors for the detection of disease markers and environmental pollutants.The specific research contents are as follows:（1）A ratio sensor for visual detection of human immunodeficiency virus gene was constructed based on the C-Ag⁺-C hairpin probe and targe-induced catalytic hairpin self-assembly strategyRelying on the specific coordination of Ag⁺and mismatched cytosine-cytosine（C-C）,the high-efficiency inhibition of urease by Ag⁺ion,and the rapid and sensitive response of phenol red to p H,a sensitive ratiometric sensor has been designed for visual detection of human immunodeficiency virus gene（HIV DNA）.This sensor utilizes the HIV DNA to open the DNA hairpin probe containing a C-Ag⁺-C structure and initiate the catalytic hairpin assembly（CHA）reaction,releasing Ag⁺to inhibit subsequent urease-catalyzed urea hydrolysis and prevent the p H of the solution from rising.Taking the absorbance ratio of phenol red at different wavelengths(A₅₅₉/A₄₃₂)as the output signal,with the aid of the CHA amplification strategy,the sensor detects HIV DNA from 10 to130 n M in a sensitive and highly selective manner with a low detection limit of 7.8 n M.In addition,this sensor can visually distinguish different concentrations of HIV DNA within a certain range and possesses a good recovery in 1%of serum samples,which will provide new ideas for biosensor design,dipstick test,blood test,and other clinical disease prevention.（2）A double-stranded DNA nanobridge was constructed based on the hairpin DNA probe to the enhance fluorescence of the crystal violet/G-quadruplex complex for detecting lead ions and crystal violetDNAzyme and G-quadruplex are purposed as universal recognition elements to achieve multi-functional signal amplification and readout,showing great potential in the design of biosensors and nanodevices.Herein,the single-stranded DNA（ss DNA）opens the dumbbell-shaped hairpin probes,the generated double-stranded DNA（ds DNA）is constructed as a nanobridge,and the released G-rich sequence is folded into a G-quadruplex village.The fluorescence of crystal violet/G-quadruplex complex villages is enhanced by the ds DNA nanobridge connections.The fluorescence intensity and the concentration of crystal violet（CV）exhibit good linearity in the range of 10 to 250 n M,which can be used for sensitive detection of CV.Additionally,combined with Pb²⁺-dependent DNAzyme,a biosensor is constructed to sensitively detect Pb²⁺.The fluorescence signal increases as the concentration of Pb²⁺increases in the concentration range of 5 n M to 25μM.The quantitative detection limit of Pb²⁺by this method is 5 n M,and it possesses good selectivity and recovery for Pb²⁺in a variety of water samples.What’s more,the sensor successfully creates logic AND gate by controlling the inputs of Pb²⁺and CV.More importantly,the platform can sensitively detect various metal ions only by changing the DNAzyme sequence,which has the potential to be widely used in environmental monitoring.（3）Fluorometric and resonance Rayleigh scattering dual-mode biosensor for determination of the activity of alkaline phosphatase based on the cobalt（II）-dependent DNAzyme probe and Co OOH nanoflakesThe Co（II）-dependent DNAzyme probe can assist nucleic acid signal amplification,the Co OOH nanoflakes have high resonance Rayleigh scattering activity and can strongly adsorb ss DNA.A fluorometric and resonance Rayleigh scattering dual-mode sensor was constructed using Co²⁺-DNAzyme probes and Co OOH nanoflakes for detection of the activity and inhibition of alkaline phosphatase（ALP）.ALP specifically catalyzes the hydrolysis of ascorbic acid-2-phosphate to produce ascorbic acid which reduces Co OOH nanoflakes to Co²⁺ion.The Co²⁺-DNAzyme is then activated by Co²⁺ion so that the substrate strand labeled with the fluorophore and a quencher is cleaved.As a result,the fluorescent group and the quenching group are far away from each other,the fluorescence signal at 518 nm is restored,and the Rayleigh scattering intensity of Co OOH nanoflakes at 405 nm decreases.In fluorescence mode,the method works on the 0.2 to 2000 U L^-1ALP activity range,and the detection limit is 0.05 U L^-1.Besides,the method was used to validate the mechanism of the action of two classical ALP inhibitors（EDTA and Na₃VO₄）.Conceivably,it can also be applied to screen for ALP inhibitors.