Research On The Synthesis Of Silver Nanoclusters And Deveopment Of Detection Methods Based On Enzyme-assited Isothermal Amplification

DNA templated silver nanoclusters(DNA-AgNCs)can be simplely synthesized,and it is widely studied due to the small size,high quantum yield,strong photobleaching resistance,good biocompatibility,and adjustable fluorescence spectrum.The application of DNA-AgNCs in the detection of foodborne pathogens,viruses,toxins,and pesticide residues has been widely reported in recent years.However,as the mechanism of DNA template sequence and structure influencing the fluorescence properties of AgNCs has not been clearly elucidated,the synthesis of AgNCs is somewhat blind,and the high quantum yield of DNA-AgNCs is rarely reported,which limits its application to some extent.This paper studied the effect of base sequence and secondary structure of DNA template on the fluorescence property of DNA-AgNCs,explored the rule of their correlation,and obtained DNA-AgNCs with high quantum yield.Secondly,the high quantum yield DNA-AgNCs was used as the signal output,and two detection schemes were designed with the help of enzyme-assisted isothermal signal amplification technology,and established highly specific and sensitive nucleic acid detection methods.The main research contents of this paper are as follows:(1)Research on the synthesis of Hairpin-AgNCs were carried out to study the synthesis conditions such as buffer solution,pH,silver nitrate concentration,sodium borohydride concentration,reaction time,as well as the AT/GC content and distribution in the stem and the effect of stem length on fluorescence of Hairpin-AgNCs.Data showed that with the increase of the GC base,emission wavelength of AgNCs had a redshift.In this process,the wavelength of single fluorescence emission was changed to double fluorescence emission.pH and the concentration of silver ions also have synergy effect to the wavelength of AgNCs.High pH and high concentration of silver ions were favorable for the formation of red AgNCs,the proportion of GC bases on fluorescence emission wavelength had a decisive role.Specific base distribution contributed to the production of strong fluorescent AgNCs.The fluorescence of AgNCs increased with the increase of stem length in a certain range.(2)To study the effect of DNA spatial structure on the fluorescence properties of AgNCs,AgNCs was synthesized using a "bulb" shaped DNA structure composed of three single strands of DNA.In the experiment,DNA "bulb" with random sequence was used to synthesize the AgNCs with high fluorescence brightness(QY=17.2%),which showed larger size(average diameter=3.36nm)and excellent stability(96.6% after one week),and proved the decisive role of structure on the formation of AgNCs.When the "filament" and "bulb shell" were replaced,compared with the "filament",the AgNCs synthesized by DNA "bulb" showed significant effect of structure enhanced the fluorescence.Sequence studies showedthat the peak wavelength of AgNCs synthesized by "bulb" was correlated with the proportion of C base in the "filament".The larger the proportion of C,the larger the emission wavelength,and the AgNCs showed a trend of fluorescence enhancement with the increase of the number of C base.(3)A DNA detection scheme was designed based on the Hairpin-AgNCs with a high quantum yield and the principle of the amplification detection of exoenzyme assisted circulation.In this scheme,the three-way junction structure was creatively used to assist the exonuclease III digestion,the enzyme digestion efficiency could be increased up to 9 times.At the same time,only a single exonuclease III without a specific identification site can realize the target chain circulation and amplify the detection signal.This method had good selectivity in DNA detection,with a linear range of 5.0 nmol/L to 50.0 nmol/L and a detection limit of 2.8 nmol/L.(4)Based on the chain replacement isothermal amplification(SDA)model and the existing literature,a circulating isothermal signal amplification technique was proposed,and a highly sensitive fluorescence detection method for RNA was established by using the high quantum yield Hairpin-AgNCs obtained by self-screening.The two-stage chain replacement isothermal amplification cycle was designed in this technique,which greatly increased the amplification factor.The results showed that the target chain was added to induce an efficient nucleic acid amplification process.Finally,the fluorescence signal of Hairpin-AgNCs was significantly reduced due to the damage of the template structure under the action of the polymerase.The linear range was 0.02 nmol/L~1.0 nmol/L,the detection limit was 17.8pmol/L,and the recovery was 82.1%~106.6%.