Visual Recognition Of Protein Biomarkers Using A Strip Biosensor In Biocomputing Logic Operations

The advantages of proteins as a class include enormous diversity and secretion into blood and bodily fluids increases the chances to identify a marker or a panel of markers for each disease state.For example,as human coagulant terminal enzyme,thrombin plays a key role in the cascade reaction.Mainly exist in the rectum and colon cancer tissue as well as embryo on the intestinal mucosa,carcinoembryonic antigen detection is of great significance in diagnosis of cancer and understanding the treatment effect of tumor clinical.The excessive expression of mucoprotein in adenocarcinoma and eye-related disease is one of the reference on diagnosis.The development of lateral flow strip biosensors?LFB?for sensitive point-of-care testing?POCT?of cancer markers for early cancer diagnosis and treatment is a crucial need,primarily in resource-limited settings because they require low volumes of reagents and are easy to use,store and transport.The good chemical stability,easy surface modification,high extinction coefficients and distance-dependent optical properties of gold nanoparticles?AuNPs?and the oligonucleotide aptamer with high affinity and specificity of variant structure to bind target play a backbone role in building a strip sensor.Moreover,to develop an independent or connected logic system that is simple in design,not limited to solution-based applications,and involves a reliable and convenient readout would be highly desirable as it provides the ideal prediction and preciseness of testing protein biomarkers.The whole paper contains the following four parts:?1?A new class of strip biosensors has been established based on thrombin aptamer-linked gold nanoparticle well-distributed aggregates,which will occur cracking reaction when the target recognized its homologous aptamer.Combined the aptamer-cleavage reaction with the enzyme catalytic amplification system,our proposed lateral flow strip biosensor?LFB?is capable of visually detecting 6.4 pM of thrombin without instrumentation within 12 minutes.Under the optimal conditions,quantitative detection of thrombin by a portable strip reader exhibited a linear relationship between the peak area and the concentration of thrombin in the range of 6.4 pM-500 nM with a detection limit of 4.9 pM,which is three orders of magnitude lower than that of aptamer-functionalized gold nanoparticles-based LFB?2.5nM,Xu et al.Anal.Chem.2009,81,669-675?.As the aptamers have no special requirements and the gold nanoparticles can also be replaced by other metallic nanoparticles,this method for strip sensing is expected to be generally applicable in point of care testing,home testing,medical diagnostics,clinical diagnosis,and environmental monitoring.?2?Combining the specific molecular recognition reaction between biotin and streptavidin with a dual-class signal amplification system,containing the unique optical properties of the gold nanoparticles,and the catalytic reaction of horseradish peroxidase,we firstly designed a double-enhanced strip biosensor?DSB?.Qualitative detection of protein biomarkers in human serum can be realized by observing the colour of the test zone with naked eye,however,for quantitative measurements,the optical intensity of the red band was recorded using a portable strip reader.Under optimal conditions,the DSB could measure 1.0×10^-5 ng/mL CEA based on visual monitoring.The results exhibited a linear relationship between the peak area and the concentration of CEA,MUC1,TB:5.0 × 10^-6 ng/mL-20 ng/mL?2.9 fg/mL,R²=0.9852?;4.8 ng/mL-1.0× 10³ ng/mL?1.2 ng/mL,R²=0.9991?and 1.9×10^-2 nM-8.0×10²nM(1.8×10^-2nM,R²=0.9998)for quantitative detection,which had obtained the same results as the standard method?chemiluminescence?.Therefore,these results made it abundantly clear that the principle of DSB is promising for rapid identification of other types of proteins,such as ferritin,albumin and haemoglobin,as well as other analytes,such as nucleic acids and viruses on site.?3?We developed a strip biosensors array based on aptamer-modified gold nanoparticles as receptors and combined the protein-aptamer binding reaction with the streptavidin-biotin interaction as well as the sandwich format.We found that a series of protein receptors obtained a distinct response pattern to each target protein.Three proteins have been well distinguished with the naked eyes and a portable reader without mutual interference,accompanying with lower limit of detection and wider linear range.A complete set of four elementary logic gates?AND;OR;INH;NAND?and eight combinative logic gates?AND-OR;AND-INH;OR-INH;INH-NAND;AND-OR-INH;AND-INH-NAND;OR-INH-NAND;AND-OR-INH-NAND?are thoroughly realized using this array,which could eventually be applicable to the keypad-lock system with enhanced complexity in the near future.Moreover,this array shows excellent linear relationships,anti-interference capability,real human serum samples applicability,long-term storage stability and reproducibility.All indicate that this design has very good prospects for development.