Chemiluminescence Imaging for a Protein Assay via Proximity-Dependent DNAzyme Formation

Abstract

An array-based chemiluminescence (CL) imaging method is presented for simple and high throughput detection of protein targets via the formation of a proximity-dependent DNAzyme to produce sensitive CL signal. The protein array is prepared by covalently immobilizing single-stranded guanine-rich nucleic acid 1-labeled antibody 1 (GDNA1-Ab1) or GDNA-thrombin aptamer subunit 1 (Apt-P1) as the capture probe on each sensing site on an aldehyde-functionalized disposable glass chip. In the presence of target protein, hemin, and another GDNA2-Ab2 or Apt-P2 probe, a sandwich complex among the protein and two probes can be formed to trigger the proximity assembly of GDNA1, hemin, and GDNA2, which leads to the formation of hemin-G-quadruplex DNAzyme. At different sensing sites, the DNAzyme-induced CL signals are simultaneously collected by a charge-coupled device for imaging readout of the sensing events. As a proof of concept, the proposed array-based CL imaging strategy is applied to detect carcinoembryonic antigen and thrombin and shows wide linear ranges over 4 and 5 orders of magnitude with the detection limits of 0.15 ng mL(-1) and 0.49 pM, respectively. Benefiting from the one-step proximity-dependent DNAzyme formation, the assay method is extremely simple and can be carried out within 40 min. By using different probes, the array can be easily used to detect more protein analytes. The advantages of easy operation, short assay time, good sensitivity, and versatility make it a promising candidate for point-of-care testing and commercial application.

Signal Amplification for Highly Sensitive Immunosensing

“…This technique can be introduced into the formation of DNAzyme as catalytic nucleic acids for signal amplification. The same group [86] integrated the proximity assay and target-driven DNAzyme assembly strategy on a disposable glass chip to develop an array-based chemiluminescence imaging method. The peroxidase-mimicking DNAzyme was split into two fragments (GDNA1 and GDNA2), and each of them was conjugated with Ab or aptamer to prepare a pair of affinity probes.…”

Proximity hybridization-mediated isothermal exponential amplification for ultrasensitive electrochemical protein detection

“…Compared with natural enzymes, artificial enzymes, which have been extensively utilized for bioanalytical applications, have several merits owing to the flexible base sequences as well as nonspecific binding properties. 24,25 Among them, the G-quadruplexhemin complex, formed by hemin intercalated into the guanine-rich nucleic acid sequences, [26][27][28] can catalyze H 2 O 2 -mediated oxidation of reductive substrates and has been successfully employed for the electrochemical, 29,30 colorimetric, and chemiluminescent determination of nucleotides. [31][32][33][34][35] By combined application with hybridization chain reaction (HCR), isothermal exponential amplification reaction (EXPAR), and rolling circle amplification (RCA) strategy, G-quadruplex-hemin complexes have been successfully employed to fabricate biosensors.…”

Timing readout in paper device for quantitative point-of-use hemin/G-quadruplex DNAzyme-based bioassays

“…The sensitive, specific detection of a wide range of analytes, including metal ions (Hua et al, 2012;Li et al,2013;Liu et al, 2014;Yang et al, 2010;Wang and Irudayaraj, 2011;Wang et al, 2012;Wang et al, 2014c), small molecules (Garai-Ibabe et al, 2014;Wang et al, 2012;Wang et al, 2014g), DNAs (Wang et al, 2011;Wang et al, 2013;Wang et al, 2014a;Zhou et al, 2013), micro RNAs (Wen et al, 2012), proteins (Fu et al, 2011;He et al, 2012;Hou et al, 2014;Huang et al, 2013;Wang et al, 2014f;Tang et al, 2012;Zong et al, 2014), and bacterial pathogens (Gomez et al, 2014), implies the great potential of peroxidase-mimicking DNAzymes in broad applications ranging from medical diagnosis, environmental monitoring to food safety testing. These DNAzymes can effectively catalyze H 2 O 2 -mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Wang et al, 2012;Wang et al, 2014a;Wen et al, 2012;Tang et al,2012;Zhou et al, 2013), 3,3',5,5'-tetrazmethyl benzidinesulfate (TMB) (Yang et al, 2010), or luminol (Wang et al, 2011;Zong et al, 2014). As such, the existing quantitative hemin/G-quadruplex DNAzyme-based bioassays generally transducer recognition chemistry using several techniques, with spectrophotometry (Fu et al, 2011;…”

“…These DNAzymes can effectively catalyze H 2 O 2 -mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Wang et al, 2012;Wang et al, 2014a;Wen et al, 2012;Tang et al,2012;Zhou et al, 2013), 3,3',5,5'-tetrazmethyl benzidinesulfate (TMB) (Yang et al, 2010), or luminol (Wang et al, 2011;Zong et al, 2014). As such, the existing quantitative hemin/G-quadruplex DNAzyme-based bioassays generally transducer recognition chemistry using several techniques, with spectrophotometry (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Tang et al, 2012;Wang et al, 2012;Wang et al,2014a;Wen et al, 2012;Yang et al, 2010;Zhou et al, 2013) and chemiluminometry (Wang et al, 2011;Zong et al, 2014) being the two most common types. Other methods established recently include fluorescence (Hua et al, 2012;Liu et al, 2014), electrochemistry (Hou et al, 2014;Li et al, 2013;Wang et al, 2013;Wang et al, 2014c;Wang et al, 2014f;Wang et al, 2014g) and Raman scattering (Wang and Irudayaraj, 2011) techniques.…”