For example, ELISA microwell or membrane assays that use horseradish peroxidase (HRP) conjugated antibodies with either a fluorogenic or chemiluminescent substrate can be used in treatment for detect proteins down to femtomolar concentrations

For example, ELISA microwell or membrane assays that use horseradish peroxidase (HRP) conjugated antibodies with either a fluorogenic or chemiluminescent substrate can be used in treatment for detect proteins down to femtomolar concentrations.25, 26 These solution-based fluorescence methods Rabbit Polyclonal to ADCK3 have limited spatial resolution and therefore cannot be used in a surface microarray format. profiling of multiple protein biomarkers in blood and serum samples is a potentially powerful method for the analysis of diseases and the monitoring of subsequent therapeutic treatments.1, 2 For example, antibody arrays that can detect up to 120 serum biomarkers for the early stage detection and analysis of various cancers are currently available commercially from Whatman Inc. These antibody assays typically use a second set of fluorescently tagged antibodies for the detection of biomarker adsorption to the array elements.3, 4 Microarrays of RNA aptamers are emerging while an attractive alternative to antibody arrays for the multiplexed bioaffinity detection and recognition of protein biomarkers.5C8 Compared to antibodies, nucleic acid aptamers are KRas G12C inhibitor 4 less susceptible to irreversible denaturation, are more amenable to chemical modification, and may be recognized by (as compared to for antibodies) selection methods.9C12 Surface RNA aptamer constructions can also be reversibly deactivated from the hybridization adsorption of a complementary DNA sequence, and may be regenerated by the subsequent desorption of DNA from your aptamer array element. Surface plasmon resonance imaging has been established as one of the main optical methods for the direct detection of bioaffinity adsorption onto DNA, protein and RNA microarrays.13C18 We have recently employed SPRI for the detection of protein adsorption onto RNA aptamer microarrays down to a concentration of 10 nM.19 A detection limit of 10 nM is sufficient for the analysis of some biomarkers (e.g., 2-microglobulin and cystatin C)20; however, many important protein biomarkers are present in biological samples at much lower concentrations. For example, the signaling protein vascular endothelial growth factor (VEGF) is present in serum samples at picomolar concentrations and has been identified as a potential biomarker for rheumatoid arthritis and various KRas G12C inhibitor 4 cancers.21C24 For the detection of biomarkers at subpicomolar concentrations in biological samples, enzymatic amplification of the biosensor response is often required. For example, ELISA microwell or membrane assays that use horseradish peroxidase (HRP) conjugated antibodies with either a fluorogenic or chemiluminescent substrate can be used in treatment for detect proteins down to femtomolar concentrations.25, 26 These solution-based fluorescence methods have limited spatial resolution and therefore cannot be used in a surface microarray format. In contrast, an HRP substrate such as 3, 3, 5, 5-tetramethylbenzidine (TMB) that creates a localized surface precipitation reaction can be used in surface biosensor microarrays with high spatial resolution. This localized precipitation reaction can be recognized with either optical or electrochemical methods.27, 28 With this paper, we display that HRP conjugated antibodies can be used with SPRI measurements of RNA aptamer microarrays to detect protein biomarkers down to subpicomolar concentrations having a localized precipitation reaction. An RNA aptamer/protein biomarker/antibody-HRP sandwich structure is formed within the microarray surface, and a subsequent localized HRP-TMB precipitation reaction is used to amplify the SPRI response due to specific protein biomarker adsorption onto the RNA aptamer array. The SPRI measurements have a subpicomolar level of sensitivity; as a first example, human being thrombin protein was KRas G12C inhibitor 4 recognized at a concentration of 500 fM using an RNA aptamer recognized from a microarray of three potential thrombin aptamer candidates. The protein detection level of sensitivity of SPRI was improved by a factor of 10,000 through the use of the HRP-TMB precipitation reaction. This amplified method was then used with a second RNA aptamer array to detect the protein biomarker VEGF at a biologically relevant concentration of 1 1 pM. II. Experimental Considerations Materials 11-amino-1-undecanethiol hydrochloride (MUAM; Dojindo), sulfosuccinimidyl 4-(SPRI measurements on this microarray allowed us to identify which aptamer(s) can form the surface aptamer-hTh-antibody sandwich structure. The 1st SPRI measurement, demonstrated in Number 4a, KRas G12C inhibitor 4 was used to determine the amount of hTh adsorption onto the microarray from a 10 nM answer. All three thrombin aptamers showed some binding affinity to hTh, with the D1 aptamer becoming the highest. A second SPRI measurement, demonstrated in Number 4b, was acquired after subsequent exposure of the array to a 10 nM answer of a monoclonal antibody for hTh. A significant SPRI signal increase was observed only in the R2 array elements, indicating that the antibody could only bind to the hTh-R2 aptamer surface complex. Open in a separate window Number 4 SPRI measurements of the sequential binding of hTh and hTh antibody to a four-component microarray composed of R1, D1, R2 and RV. The pattern of the microarray is demonstrated in Number 3. (a) Surface adsorption of 10 nM hTh onto the microarray. The SPR difference image was.