Novel Highly-sensitive Modified-free LC-based Biosensor And Its Application For DNA Target Detection

The sensitive and selective detection of DNA has attracted intense interest due to its wide application.Conventional DNA detection methods,such as PCR and electrophoreis based assays,are complex and not available in field applications,because the associated technologies require costly,bulky instrumentation which must be operated by the professional staffs.The DNA biosensors are generally based on complementary base-pairing and can transform the hybridization events into detectable signals.This detection method is rapid,accessible,and convenient.Fluorescence-based microarray,the most common and commercially available DNA biosensor,is highly sensitive and high-throughout.However,this method possessed several disadvantages: the fluorescence labeling of the target DNA is complex and cost,and it can greatly influence the hybridization efficiency of probes on the substrate.Besides,there are also photobleaching and quenching effects of fluorophore.Recently,the liquid crystals(LCs)material,with high degree of shape,dielectric anisotropy,and optical anisotropy,has attracted particular attention for its application in biosensors.For instance,LCs can act as the signal amplifier and the transducer in the biosensor.The LCs-based biosensor can permit sensitive detections without complex instruments and extra electrical power,which enables the simple and robust detection away from central laboratory.Up to now,LCs-based biosensors have been successfully used for the detection of proteins,antigen-antibody,nucleic acids,bacteria,viruses and other substances.Generally,the immobilization methods of DNA probes on substrates are based on attachment of DNA which was chemically modified with appropriate linking groups.Although previous studies have shown that the chemical modification of DNA is an efficient strategy to construct the LC biosensor,there are still challenges in precisely controlling the orientation,conformation,and the grafting density of surface-tethered DNA,which would greatly influence the hybridization ability of probes,the repeatability,and even the LCs' response.Besides,the attachment of DNA chemically modified with linking groups always needs a large amount of complicated steps,which make it complex and costly,and even limit the reproduction.In our study,we combined the existed LC biosensor technologies with the novel DNA probes immobilization approach proposed by A.Opdhl and his co-workers.This novel immobilization approach was based on the stronger adsorption of adenine bases on gold surface compared with theother-three DNA bases.When multiple consecutive adenine sequence is incorporated into a DNA strand as a terminal block,it can serve as an effective anchoring block for the high-intensive preferentially chemisorption of DNA on Au.What's more,the poly A block can be easily produced with probes' recognition block due to the mature PCR technique,which eliminates the extra modification of DNA probe and reduces the synthesis cost.Moreover,our immobilization approach is based on the saturation adsorption of the probes and only needs one step.All makes it possible to precisely regulate the lateral spacing,configuration,and surface density of surface-tethered DNA by tuning the poly A length.The appended recognition block of surface-tethered DNA is upright and brush-like,which is well-suitable for the DNA hybridization.This novel LC-based DNA biosensor(or called LC cell)is mainly composed of 3 parts: silanized substrates,DNA tethered substrates,and the LCs of which the thickness is limited by a piece of 20 ? m-thick copper grid confined between two substrates.The SAM of DMOAP on the silanized substrates allows perpendicular embedding of LC molecules,and then induces the hometropic orientation of bulk LCs.Subsequently,this ensures the uniform black optical background of LC cells and provide a maximum contrast between the background and signals.Under appropriated parameters(such as probe density,DNA length,orientation,etc),surface-tethered single stranded DNA can also induce the hometropic orientation of LC withoutother alignment moleculars.After the hybirdization of surface-tethered probe DNAs with complementary chains(target DNA),the double stranded DNAs will form on the substrate.And this would greatly change the surface topology of the substrate and induce the homeotropic-to-tiled transiton of the LC molecules around them.Then,the transition is amplified into the bulk LCs film for macroscopic distances,causing changes in their effective birefringence.Ultimately,these changes are transformed into optical signal,which can be observed with bare eyes through crossed polarizer,and realize the detection of target DNA.We also explored and optimized the key factors related closely to LC cell's performances.LC cells with optimized factors were constructed for the detection of target DNA.The detection limit of the LC cells was low to0.1nM with a relatively good specificity.To enhance the sensitivity,we used the AuNPs loaded with signal probes.The Au NPs functionalized with signal probes can be bound to the free-end of target DNAs captured by probes immobilized on surface,further enhancing the surface topology changes after the target DNA hybridization.Then,the optimized LC cells were for the detection of target DNA with the LOD of 0.1pM and excellent specificity.Therefore,the highly sensitive detection of trace DNA target without complex instruments is successfully realized.