Electrogenerated chemiluminescence peptide-based bioassay

Peptide fixed on the electrode surface

A specific peptide (FYSHSFHENWPS) can selectively bind to cardiac troponin I (cTnI) with a disassociation constant of the complex in a nanomolar level (Park et al. 2010). Using the specific peptide as the molecular recognition element, we developed an ECL peptide-based biosensing method for the determination of cTnI with gold nanoparticle amplification (Shan et al. 2014). Capture peptide was self-assembled on the surface of gold nanoparticle-modified gold electrode through a thiol-containing cysteine at the end of the capture peptide to obtain ECL biosensor. When the biosensor reacted with target cTnI and then incubated with ECL probe (bis(2,2′-bipyridine)-4,4′-dicarboxybipyridine ruthenium-di(N-succinimidyl ester) [Ru(bpy)₂(dcbpy)NHS, Ru1] labeled signal peptide), a strong ECL response was electrochemically generated. The good affinity of peptide and the amplification of gold nanoparticles resulted in a sensitive detection of cTnI from 1.0×10^-12 to 3.0×10^-10 g/ml with a detection limit of 0.4 pg/ml. The peptide fixed on the electrode surface provides a simple and selective heterogeneous method for the assay of biomedical samples, especially when it contains a high concentration of interference proteins. The process is complicated because it includes immobilization and washing steps.

Homogeneous model

Another configuration is the homogeneous model. The binding of target to peptide in solution results in the change of ECL signal. One homogeneous ECL method for the determination of cTnI was reported in our group (Wang et al. 2012). In this work, the peptide (FYSHSFHENWPSK) as the molecular recognition element was labeled with ruthenium complex through NH₂-containing lysine on the peptide via acylation reaction and used as an ECL probe. In the presence of cTnI, a decrease in ECL signal was observed upon the binding event between the ECL probe and target cTnI. The larger difference between small peptide and protein in homogeneous ECL peptide-based bioassay results in a sensitive and selective determination of cTnI with a detection limit of 1.2×10^-10 g/ml. However, the binding interaction between peptide and target is limited due to the limited binding epitopes, and the ECL signal is limited because only one ECL molecule is attached directly to each peptide.

Binding interactions can be increased by the binding of multiple ligands on one entity to multiple receptors (Terskikh et al. 1997, Vance et al. 2008). Thus, the employment of polyvalent binding motif of the probe on the appropriate platform for the target protein is promising in the development of a sensitive peptide-based bioassay. We developed a homogeneous ECL peptide-based method by incorporating liposome and magnetic bead as amplification platform for the determination of cTnI (Qi et al. 2013). The principle scheme is shown in Figure 2. Liposomes were synthesized using a standard sonication procedure and used as the carrier of ECL signal reagents for signal amplification. The magnetic capture peptides were synthesized by covalently attaching the peptides to the surface of magnetic beads via acylation reaction, and the liposome peptides were synthesized by covalently attaching the peptides to the signal reagent-encapsulated liposomes. In the presence of cTnI, sandwich-type conjugates were generated in the incubation of the magnetic capture peptides and the liposome peptides. After a magnetic separation, the sandwich-type conjugates were treated with ethanol; thus, a great number of the ECL reagents were released and measured by the ECL method. The large amounts of ECL reporter molecules (1.9×10⁷ Ru1 molecules/liposome) and both large active surface areas of the liposome and magnetic beads providing polyvalent binding motifs for the target protein result in an extremely low detection limit of 4.5 pg/ml for cTnI, which opens a new door to the ultrasensitive detection of proteins in clinical analysis.

Figure 2

Schematic diagram of the liposome peptide-based ECL method for the determination of cTnI.

Reprinted with permission from Qi et al. (2013). © 2013 American Chemical Society.

Cleavage of peptide

A peptide with the amino acid sequence HSSKLQ can be used as a substrate to measure prostate-specific antigen (PSA) enzymatic activity in extracellular fluids with high degree of specificity and good stability (Denmeade et al. 1997). Several peptide-based methods were developed for the detection of PSA based on the target-induced cleavage of peptide, such as fluorescent peptide-based assay (Choi et al. 2013, Lee et al. 2013) and electrochemical peptide-based assay (Roberts and Kelley 2007, Zhao et al. 2010). We developed an ECL peptide-based method for the determination of PSA based on the target-induced cleavage of peptide (Qi et al. 2012). A specific peptide with a sequence of CHSSKLQK was labeled with Ru(bpy)₂(dcbpy)NHS and immobilized onto a gold electrode surface via a self-assembling technique to obtain the ECL-PB biosensor. The peptide substrate was recognized and cleaved by PSA, resulting in the decrease of amount of ECL emitter and then the decrease of ECL signal. The direct transduction of peptide cleavage events into an ECL signal provides a simple, sensitive method for the detection of PSA. The detection limit of 0.038 ng/ml was obtained. However, in this protocol, the ECL intensity of the biosensor decreased with the increase of PSA concentration, which is known as a “signal-off” mode. The signal-off property is undesirable because it is more susceptible to background variation and the room for signal change is also small.

Therefore, another “signal-on” ECL peptide-based biosensor for the determination of PSA by combining both signal enhancement from gold nanoparticle and signal quenching from ferrocene was fabricated recently in our laboratory (Qi et al. 2014). The specific peptide (CHSSKLQK) served as a substrate, and Ru(bpy)₃²⁺ was used as an ECL signal. The schematic diagram of the fabrication process of ECL-PB biosensor for the determination of PSA is shown in Figure 3. Gold nanoparticles were first modified onto Nafion-modified glassy carbon electrode by casting the mixture of Nafion and gold nanoparticles, and then Ru(bpy)₃²⁺ was effectively incorporated into the composite film of Nafion based on the electrostatic interaction. Then, ferrocene-labeled specific peptide was self-assembled on modified electrode. In the absence of PSA, a lower ECL signal was observed due to the quenching of the attached Fc on the ECL of Ru(bpy)₃²⁺. In the presence of PSA, PSA recognized and cleaved the peptide on the electrode surface, resulting in the decrease of the amount of Fc on the electrode surface and thus an increase of ECL signal. A signal-on ECL peptide-based method was developed for the determination of PSA via target-induced cleavage of peptide. Due to the enhancement of the gold nanoparticles on the ECL signal and the low background of ECL-PB biosensor from Fc quenching, a low detection limit of 8×10^-13 g/ml was obtained for PSA.

Figure 3

Schematic diagram of the fabrication process of ECL-PB biosensor for the determination of PSA.

Reprinted with permission from Qi et al. (2014). © 2014 American Chemical Society.

Phosphorylation of peptide

The serine, threonine, or tyrosine residues in peptide or protein can be phosphorylated by protein kinase in the presence of ATP molecule. The phosphorylation of proteins catalyzed by kinases is known as a control mechanism with most aspects of physiology functions. The measurement of kinase activities and the identification of their potential inhibitors are not only necessary for understanding many fundamental biological metabolism processes but also important for the research of cancer development, diagnosis, and treatment. Therefore, peptide can be used as the substrate to assay the activity of protein kinase. The ECL method was also employed as the detection technique to assay protein kinase activity. The generally used methods for the measurement of protein kinase activities mostly relied on phosphor-specific antibodies bound to the phosphorylated proteins specifically (Figure 4A). For example, Xiao et al. (2007) developed a sensitive ECL method for the detection of protein tyrosine kinases with 240 fmol/well of the kinase reaction product using ruthenium derivative-labeled monoclonal antibody against phosphotyrosine. Other ECL strategies were also proposed to measure protein kinase activities, including employing adenosine 59-[c-thio] triphosphate (ATP-s) as cosubstrate and gold nanoprobe as ECL signal (Figure 4B) or employing zirconium cations (Zr⁴⁺) as linking agent to recognize phosphates and rapidly link two biomolecules containing phosphate groups (Figure 4C).

Figure 4

Scheme of possible configurations of the ECL peptide-based bioassay for the detection of protein kinase.

(A) sandwich immunoassay; (B) ATP-s as cosubstrate and gold nanoprobe as ECL signal; (C) Zr⁴⁺ cations as the linking agent.

Xu et al. (2010) reported an ECL biosensor using gold nanoparticles as signal transduction probes for the kinase activity and inhibition analysis. The schematic representation of ECL strategy for kinase activity detection is shown in Figure 5. Kemptides (CLRRASLG) were self-assembled on the gold electrode to obtain peptide-modified electrode. After the phosphorylation reaction in the presence of protein kinase A (PKA) using ATP-s as the cosubstrate, thiolated substrate peptides were introduced on the electrode surface. Then, gold nanoparticles were specifically conjugated to the thiophosphate group. Based on the catalysis of gold nanoparticles to the luminol ECL reaction, PKA was measured in the range of 0.07–32 U/ml with a low detection limit of 0.07 U/ml. The method was also used to quantitatively evaluate the inhibition of kinase in the presence of small molecule.

Figure 5

Schematic representation of ECL strategy for kinase activity detection using gold nanoparticles as signal transduction probes. Reprinted with permission from Xu et al. (2010). © 2010 American Chemical Society.

Since then, various nanoparticles, including gold nanoparticle and silica nanoparticles, with different strategies have been used in the ECL peptide-based bioassay for the determination of protein kinase. For example, Zhao et al. (2012) used DNA-modified gold nanoparticles that can absorb a great deal of Ru(II) by electrostatic interaction to develop the ECL method for the determination of protein kinase and inhibition. First, an ECL nanoprobe was prepared by conjugating gold nanoparticles with phosphorylated DNA capture probes and TBR-cysteamine. In the presence of PKA and ATP, substrate biotinylated peptides were phosphorylated and then conjugated to streptavidin-coated magnetic beads via the biotin-streptavidin interaction. Zr⁴⁺ cations are used for linking ECL nanoprobes with peptides and result in the capture of ECL nanoprobes on the surface of magnetic beads. The complexes were then captured and enriched on the electrode surface for ECL detection. The linear range was from 0.01 to 50 U/ml and the detection limit of PKA is 0.005 U/ml.

Chen et al. (2013) developed an ECL method for the determination of PKA and inhibition by using linking agent of Zr⁴⁺ cations and signal amplification of Ru(II) encapsulated phosphorylate-terminated silica nanoparticles (R-PSiNPs). R-PSiNPs were prepared by the controlled synchronous hydrolysis of tetraethoxysilane and 3-(trihydroxysilyl)-propylmethyl-phosphonate in water-in-oil microemulsion. Peptide (CLRRASIG) is self-assembled on the gold electrode surface via Au-S bonding and then phosphorylated by PKA in the presence of ATP. R-PSiNPs were subsequently grafted to electrode surface by linking with phosphorylated peptide in the presence of Zr⁴⁺. The method can be employed to assay PKA activity with a detection limit of 0.005 U/ml.

Liang et al. (2014) reported an ECL biosensor for the determination of protein kinases and inhibition using enzyme-functionalized gold nanoparticles as amplification platform. Biotin-DNA and glucose oxidase (GOx) were first conjugated to gold nanoparticles to form GOx/AuNPs/DNA-biotin nanoprobe. Peptides were first assembled onto gold electrode surface and then phosphorylated by PKA in the presence of TPA. The assembled phosphorylated peptides are subsequently recognized by biotinylated anti-phosphoserine antibody. Then, the nanoprobe was bound to the biotinylated anti-phosphoserine antibody on electrode surface through a biotin-avidin interaction. GOx assembled on the nanoprobe can catalyze glucose to generate H₂O₂ in the presence of O₂, whereas the ECL reaction occurred in the luminol system. This biosensor can also be used for the quantitative analysis of ellagic acid.