Aptamer-Based Biosensors for the Colorimetric Detection of Blood Biomarkers: Paving the Way to Clinical Laboratory Testing

Abstract

Clinical diagnostics for human diseases rely largely on enzyme immunoassays for the detection of blood biomarkers. Nevertheless, antibody-based test systems have a number of shortcomings that have stimulated a search for alternative diagnostic assays. Oligonucleotide aptamers are now considered as promising molecular recognizing elements for biosensors (aptasensors) due to their high affinity and specificity of target binding. At the moment, a huge variety of aptasensors have been engineered for the detection of various analytes, especially disease biomarkers. However, despite their great potential and excellent characteristics in model systems, only a few of these aptamer-based assays have been translated into practice as diagnostic kits. Here, we will review the current progress in the engineering of aptamer-based colorimetric assays as the most suitable format for clinical lab diagnostics. In particular, we will focus on aptasensors for the detection of blood biomarkers of cardiovascular, malignant, and neurodegenerative diseases along with common inflammation biomarkers. We will also analyze the main obstacles that have to be overcome before aptamer test systems can become tantamount to ELISA for clinical diagnosis purposes.

Keywords: aptasensors; blood biomarkers; colorimetric detection; point-of-care testing.

Figure 1

Colorimetric detection using the dispersion/aggregation of gold nanoparticles (AuNPs) in salted solution. Unmodified AuNPs aggregate in the salt-containing solution, turning a red colored solution into a blue solution. The non-specific absorption of nucleic acids prevents the aggregation of AuNPs, and the solution remains red.

Figure 2

General scheme of detection for a colorimetric aptasensor with peroxidase (A) or peroxidase-like (B) generation of the analytical signal. First, a biotinylated aptamer forms a complex with the analyte in a microplate well; then, streptavidin-conjugated peroxidase binds biotin. Next, peroxidase (A) or a peroxidase-mimicking analog (B) oxidizes the chromogenic substrate, turning a colorless solution into a colored solution.

Figure 3

Chemiluminescent VEGF detection based on the peroxidase-mimicking activity of the hemin and G-quadruplex aptamer complex [17]. Target binding induces quadruplex structure formation in the VEGF aptamer. The resulting aptamer–target complex binds hemin and catalyzes the oxidation of luminol in the presence of hydrogen peroxide.

Figure 4

AuNP-based aptasensor for VEGF detection with signal amplification proposed by C.C. Chang [19]. The aptasensor consists of aptamer-containing hairpin DNA, two DNA substrates, and two auxiliary DNA fragments. Without the target, the single-stranded auxiliary DNA fragments are adsorbed on the AuNPs, preventing their aggregation and giving a red color to the solution. The addition of VEGF switches the aptamer to an active structure, which leads to the reorganization of the hairpin DNA. “Opened” hairpin DNA, in turn, forms a duplex with the DNA substrate and initiates a nonlinear chain reaction involving the auxiliary DNA fragments. The resulting dendrimer-like structure is poorly adsorbed on the AuNPs, and their aggregation causes a red to blue color change.

Figure 5

Multicolor aptamer-based system for CD63-positive exosome detection [21]. Exosomes are captured by CD63-specific aptamers immobilized on magnetic beads. Then, a cholesterol-modified DNA anchor embeds into the lipid bilayer of exosomes, with the ssDNA “sticky” end exposed to trigger a chain hybridization reaction with the biotinylated oligonucleotides H1 and H2. Next, H1 and H2 bind with streptavidin-conjugated alkaline phosphatase. The dephosphorylation of ascorbic acid phosphate in silver salt solution leads to the deposition of a silver shell on the surface of the AuNRs and a resulting multicolor change.

Figure 6

Lateral flow assays for CD63-positive exosomes. (A) Au@Pd nanoparticle-based aptasensor proposed in [25]. An anchor DNA fragment conjugated with Au@Pd nanopopcorn forms a complex with exosomes. Nanoflower-modified CD63 aptamers provide exosome concentration at the test line. Subsequent laser irradiation generates a thermal signal and produces a characteristic black band at the test line. (B) AuNP-based aptasensor developed in [26]. Without exosomes, the aptamer conjugated with AuNP binds to a complementary DNA fragment at the test line, producing a colored band due to the accumulation of AuNPs. In the presence of exosomes, the AuNP-modified aptamer binds CD63 on the exosome surface and the test line remains colorless.

Figure 7

Lateral flow assay for dopamine detection proposed in [41]. Without dopamine, the aptamer forms a red-colored complex with AuNP-modified DNA1, which is trapped by DNA2 at the control line. In the presence of dopamine, the aptamer dissociates from AuNP-modified DNA1, and duplex formation between DNA1 and DNA3 provides red coloring at the test line.

Figure 8

Aptamer-based lateral flow assay for cortisol detection developed in [45]. In the presence of cortisol, the aptamer binds to its target, while unbound AuNPs interact with membrane-bound cysteamine, resulting in red line formation. Without cortisol, the aptamer is adsorbed on AuNPs and prevents their interaction with cysteamine; thus, the membrane remains colorless.

Figure 9

Aptamer-based system for thrombospondin-1 detection developed by K. Ji et al. [54]. The aptamer on magnetic beads forms a duplex with biotinylated DNA that binds the streptavidin–HRP conjugate. HRP oxidizes the chromogenic substrate and generates a colorimetric signal. Throbmospondin-1 displaces biotinylated DNA from the complex with the bead-bound aptamer. The peroxidase conjugate cannot bind with the beads, which leads to a decrease in the colorimetric signal intensity.

Figure 10

Insulin detection by aptasensor based on Au nanorods with peroxidase-like activity [59]. Without insulin, AuNRs catalyze the oxidation of the chromogenic substrate (TMB) in a peroxidase-like manner, resulting in a color change in the solution. Insulin binds with the aptamer on the AuNRs and inhibits the oxidation of TMB.

Figure 11

Chemiluminescent detection of RBP4 proposed in [61]. RBP4-specific aptamer captures the analyte in the microplate well. The aptamer–RBP4 complexes are visualized via conjugates of anti-RBP4 antibodies with covalently crosslinked luminol-modified AuNPs.

Figure 12

Lateral flow assay for vaspin detection based on two aptamers [62]. The complex of vaspin and AuNP-modified aptamer 1 binds with aptamer 2 at the test line, resulting in an increase in AuNP concentration and red coloring. In the absence of vaspin, AuNP-aptamer 1 binds the complementary DNA and provides red coloring at the control zone.