General Colorimetric Detection of Proteins and Small Molecules Based on Cyclic Enzymatic Signal Amplification and Hairpin Aptamer Probe

Abstract

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

“…Thus, to make the detection of trace levels of the analyte possible, many researchers have devoted effort to developing amplification techniques. Signal amplification strategies, such as the polymerase chain reaction (PCR) [5][6][7], rolling circle amplification (RCA) [8][9][10][11], enzyme-assisted signal amplification [12][13][14] and DNA based technologies [15][16][17][18] have been employed to improve the sensitivity of protein detection. Meanwhile, a further development in DNA amplification is the hybridization chain reaction (HCR) [19], which is enzyme-free and needs initiator DNA to trigger a self-assembly process.…”

Binding-induced collapse of DNA nano-assembly for naked-eye detection of ATP with plasmonic gold nanoparticles

“…Recently, localized surface plasmon coupling of gold nanoparticles (AuNPs) has been widely employed to develop colorimetric detections. Especially, unmodified AuNPs based methods have attracted extensive interests because of their easy operation and simple readout (Arvizo et al, 2013;Du et al, 2011;Li and Rothberg, 2004;Li et al, 2012a;Liu et al, 2013;Liu et al, 2012;Nelson and Rothberg, 2011;Sener et al, 2014;Wang et al, 2007;Xia et al, 2010;Zhang et al, 2013;Zhang et al, 2012). The unmodified AuNPs can effectively discriminate double stranded or folded DNA from flexible, single stranded DNA, which leads to an obvious color change from red to purple or blue (Akhlaghi et al, 2012;Li and Rothberg, 2004;Nelson and Rothberg, 2011;Zhang et al, 2012).…”

“…The unmodified AuNPs can effectively discriminate double stranded or folded DNA from flexible, single stranded DNA, which leads to an obvious color change from red to purple or blue (Akhlaghi et al, 2012;Li and Rothberg, 2004;Nelson and Rothberg, 2011;Zhang et al, 2012). Based on this principle, aptamers are widely used as ligands to bind small molecules and the resulting conformational change of aptamer upon binding can be discriminated by the color change of unmodified AuNPs (Arvizo et al, 2013;Du et al, 2011;Li and Rothberg, 2004;Li et al, 2012a;Liu et al, 2013;Liu et al, 2012;Nelson and Rothberg, 2011;Sener et al, 2014;Wang et al, 2007;Xia et al, 2010;Zhang et al, 2013;Zhang et al, 2012). The above mentioned prototype detection platform were successfully used to detect small molecules such as ATP, cocaine and glucose as low as sub-micromole.…”

Aptamer‐based detection and quantitative analysis of human immunoglobulin E in capillary electrophoresis with chemiluminescence detection

“…Zhao et al [31] constructed a gold nanoparticles (AuNPs) sensing platform for detection of thrombin by chemiluminescence resonance energy transfer method with a LOD of 15 pM. A colorimetric method for detection of thrombin and adenosine triphosphate (ATP) was also described by Li et al based on cyclic enzymatic signal amplification and hairpin aptamer probe [32]. The LODs of thrombin and ATP are 50 pM and 100 nM, respectively.…”