Protein Detection Using Apatamer-Based PSi Optical Sensors

Thrombin is a central protease for hemostasis and thrombogenesis in the human body,and thrombin level in the blood is the key to evaluating the tendency of thrombosis and the coagulation function in clinical diagnosis.However,the methods of detecting thrombin clinically can only reveal the level of generated thrombin indirectly now.There are also quite a few requirements for both operators and patients and these methods are susceptible to interference.Therefore,there is an urgent need for a rapid and simple detection method for thrombin in clinic diagnosis.Nowadays,immunoassays which are based on antigen-antibody interaction,are still mainstream approaches to measuring thrombin.But in recent years,aptamers are employed increasingly in thrombin biosensing as alternatives to antibodies.Because aptamers not only own high binding affinity to target molecules in analogy with antibodies,but also have more unique features,such as small size,low molecular weight,easy fabrication,versatile surface modification,sequence tunability,reversible folding,excellent stability and so on.Consequently,aptamer-based biosensors are expected to be applied in thrombin detection for clinic samples.Nanopore technology provides a new strategy for high-throughput and highly sensitive detection of biological marcromolecules like proteins.When a protein molecule passes through a nanopore of similar size,there will be a sharp change in the optical properties of the nanopore,and this change is closely related to protein properties.Based on this principle,open-ended porous silicon(OEPSi)functionalized with aptamers was utilized as the biological recognition element for thrombin detection with reflection interference spectroscopy(RIFS)as the transducing strategy in this study.All work is present in the following:(1)The research progress of biosensors using aptamers as probes is summarized,and aptasensors in protein detection is highlighted in this section.Besides,research advance in solid-state nanopore(SSN)sensing technology and aptamer-modified SSN sensing is present in this chapter.PSi is a novel SSN material and often functions as the sensitive material in RIFS biosensors.Thus,the application of PSi biosensors including those with oligonucleotide probes is also concluded.(2)A thrombin detection biosensor was fabricated with thrombin-binding aptamers(TBA)as capture probes and OEPSi as the sensing element for RIFS.In the proposed sensor,TB A was immobilized on the inner walls of PSi nanochannels through chemistry modification.So,thrombin in sample solution would be captured by TBA when flowing through PSi channels.Once thrombin is trapped in the PSi layer,there will be an increase in the average refractive index causing redshifts in the reflection spectra and changes of effective optical thickness(EOT).Based on this mechanism,a label-free optical biosensor for thrombin detection with high sensitivity,high selectivity,wide detection range,low detection limitation and regeneration abitlity was fabricated.In comparison with closed-ended PSi sensors,the response rate(<2h)and sensitivity are higher in this flow-through configuration.It is also found that solution environment(such as pH,ionic strength,etc.),pore size and aptamers can alter the capture ability of aptamers.(3)Based on the sensing principle that last chapter mentioned,thrombin detection in clinic samples was realized through sequence design of aptamers with the linear detection range from 10 to 1000 nM and the detection limitation at nanomolar scale.So,this biosensor has an enormous potential in clinical diagnosis.It is also seen there is no obvious difference in the calibration curves between buffer and serum,which indicates this sensor is free of background interference.For thrombin detection in serum,the recovery rates maintained between 90%and 110%with high accuracy(RSD<10%).And there is no significant difference between the proposed strategy and ELISA assays,verifying the reasonability of the designed OEPSi sensor.Besides,a dual binding-site PSi sensor was developed and we found that the sensitivity can be further improved by increasing the thrombin binding-sites.