5.1. Direct Fluorescent Labeling
The detection mechanism of this class of assays is based on a fluorescent label either directly conjugated to the detection antibody or subsequently added to the detection antibody after the binding event. Thus, for this class of assays, the fluorescence signal of antigen–antibody binding event is limited to that of the label directly conjugated to the antibody. For example, a study reported by Jiang et al. [28
] described a sandwich immunoassay for cTnI, captured between an antibody immobilized on magnetic nanoparticles and a biotin-conjugated detection antibody. The fluorescent signal of streptavidin-conjugated quantum dots (QDs) is used to signal the presence of cTnI upon binding to biotin. The detection limit of the assay was 0.047 ng/mL and with a range of 0–40 ng/mL. While this assay is sensitive enough to detect abnormal levels (>5 ng/mL) of cTnI, its LDR is too narrow to reflect the extent of MI, which produces much higher concentrations of cTnI in the patient’s blood.
Another example of an extended type of sandwich cTnI immunoassay uses three-dimensional (3D) nanotube arrays of TiO2
to immobilize a polyclonal anti-troponin antibody where cTnI is captured. This 3D immobilization platform increases the concentration of the capture antibody on the surface, which helps to lower the LOD of the assay. Following the antigen–primary antibody interaction, there is the binding with a secondary antibody that, subsequently, binds to a fluorophore-labeled (AM700) tertiary antibody. The fluorescence emission is then proportional to cTnI levels that could be detected at concentrations as low as 0.1 pg/mL [31
], however, the LDR of the assay 0.1–1 ng/mL. This LDR makes the assay not relevant for quantification of cTnI at clinical applications unless the samples undergo a series of dilutions.
A similar approach of signal amplification based on the fluorescent of Alexa Fluor-conjugated detection was reported recently for lateral flow immunoassay (LFIA). The capturing antibody is immobilized on cellulose nanofibers (CNFs) over a paper platform where CNFs help increase the number of capture antibodies on the surfaces (similar to 3D TiO2
nanotubes above). This enhances the sensitivity of this sandwich immunoassay for visual detection of the fluorescent signal. However, the LDR of 2.10–2.75 ng/mL renders the assay technically not useful for practical applications in clinical settings [43
Lou et al. introduced a similar example of lateral flow immunoassay (LFIA) but the detection antibody and the fluorescent label are conjugated to a nanosphere [29
]. The nanosphere is layered with biotinylated BSA, followed by fluorescent-labeled (Alexa Fluor 647) streptavidin, and then by biotinylated detection antibody. The lateral flow immunoassay (LFIA) starts by capturing cTnI on a nitrocellulose pad coated with capturing antibody. The functionalized labeled nanospheres are then accumulated on the antigen and, hence, the fluorescent signal intensity is enhanced. The LFIA exhibited high cTnI detection sensitivity (LOD of 0.049 ng/mL) with LDR of 0.049–50.0 ng/mL. This detection range enables the application of this assay into the clinical test but it may require dilutions for a blood sample that has high levels of cTnI.
Liu and co-researchers introduced a simple cTnI quantifying fluorescent assaybased on the competitive binding of the fluorescent-labeled aptamer to cTnI vs. graphene oxide (GO) surface. This assay is not a sandwich immunoassay and it does not require the use of any antibodies or immobilizing surface for capturing the antigen. Instead, in the absence of the protein, the aptasensor labeled with 6-carboxyfluorescein (6-FAM) binds to the GO surface, which quenches its fluorescence. Upon introducing cTnI, the aptamer binds to the protein preferably and separates from the GO surface, which turns on the fluorescence of 6-FAM. The enhancement of the fluorescence intensity in correlation to cTnI levels is the key detection mode. This novel assay exhibited high selectivity to cTnI compared with other interference proteins (HSA, BSA, IgA, IgG, and AFP) in the range of 0.10–6.0 ng/mL and a low detection limit of 0.07 ng/mL [30
]. Although this assay is simple and quick to apply with high selectivity, the narrow LDR limits its practical applications.
5.2. Package of Fluorescent Labels
On the other hand, Järvenpää et al. reported a single-step two-site cTnI sandwich immunoassay where europium(III)-chelate-dyed nanoparticles are used as the fluorescent label [33
]. This strategy amplifies the signal for each antibody–antigen binding event because the concentration of the fluorescent label on the surface of the nanoparticle is much larger than the concentration of the label directly conjugated to the detection antibody (as in (i) above). The assay employs streptavidin-coated microtiter wells immobilized with a biotinylated monoclonal antibody and an enzymatically digested F(ab’)2 antibody. The quantification of cTnI is then attained by the simultaneous addition of the cTnI sample and the nanoparticles-conjugated capture antibody followed by measuring the fluorescence of the europium-bound nanoparticle (also known as “lanthanide luminescence”). The assay has an LOD of 0.0020 ng/mL, a limit of quantification of 0.012 ng/mL, and an LDR of 0.003–9.6 ng/mL; this detection range is not practical for clinical settings.
Another way of increasing the concentration of fluorescent labels per binding event is by introducing a large load of fluorescent dyes. This was achieved by Wang et al. using coumarin (COU)-loaded metal-organic frameworks (MOFs), which have a high loading capacity of molecules because of their high porosity and surface area. Hence, the detection of cTnI by an antibody conjugated to a COU-loaded ZIF-8 MOFs ([email protected]
/Ab) amplifies the detection signal via the release of alkaline-hydrolyzed COU (green-blue fluorescence) from the MOFs’ interior (Scheme 2
). The fluorescence intensity is quantitatively related to the amount of cTnI captured providing a wide dynamic detection range of 0.026 ng/mL to 0.85 ng/mL with an LOD of 0.1 ng/mL [32
]. This assay is then not suitable for the measurement of cTnI patients with a series of dilutions.
A more recent example of fluorescent–loaded anti-cTnI was reported by He and coworkers where acridinium ester-loaded into poly[(N
-isopropyl acryl-amide)-co-(methacrylic acid)] (P(NIPAM-co-MAA)) microspheres were conjugated to detection antibody [44
]. The capture antibody is immobilized on magnetic fluorescent nanobeads. After cTnI is sandwiched between the capturing and detecting antibodies, the solution is heated so that the acridinium ester is released from the microspheres and hydrolyzed to produce a chemiluminescent signal (Scheme 3
) in addition to the fluorescent signal of the magnetic nanobeads. The LOD of 0.116 pg/mL and an LDR of 0.1–40 ng/mL, which are relevant for the clinical settings.
Along the line of increasing fluorescent label density per binding event, Li et al. reported [34
] a developed form of his aptamer assay by using a sandwich immunoassay whereby the detection antibody is replaced by aptamer-primer with a padlock probe for rolling circle amplification (RCA). The assay uses a simple microplate antibody–antigen reaction followed by RCA to generate a complementary strand for FAM nucleotide. The fluorescent probe is then released from the surface of graphene oxide and hybridized with the RCA product leading to amplified enhancement of the fluorescent signal with a detection limit as low as 14.40 pg/mL and an LDR range of 0.050–0.5 ng/mL [34
In 2020, the same researchers developed an aptamer-based immunoassay coupled with a molecular beacon fluorescent probe and rolling circle amplification (RCA) for the accurate and sensitive detection of cTnI. In this strategy, the aptamer can act as a communication bridge between proteins and oligonucleotides. RCA amplification produces a circular template which is detected by fluorescence molecular beacon probe. This new method provides the specific and sensitive determination of cTnI with a limit of detection of 7.24 pg/mL [45
5.3. Enzyme-Linked Assays
Enzyme-linked immunoassays are based on signal amplification by the catalytic activity of the enzyme to produce fluorescent products. Hence, each antibody–antigen binding event by enzyme-conjugated antibody produces a high concentration of the fluorescent label (even larger than that of (ii) above). The concentration of the antigen is then reflected through the generated fluorescent signal of the product. The disadvantage of this amplification process, however, is the need to monitor the signal over a specific (short) period. Otherwise, even with low concentrations of antigen, the signal can still saturate due to the high catalytic activity of the enzyme.
Liu and coworkers reported the use of alkaline phosphatase (ALP) to produce the non-fluorescent p
-aminophenol which upon reaction with ethylene diamine produces fluorescent polymer carbon dots to detect cTnI at a wide concentration range with LDR of 1.0–30.0 ng/mL and an LOD of 1.0 ng/mL [37
]. A similar strategy for using ALP was also reported by Zhao et al. [35
] where ALP-mediated hydrolysis of m
-hydroxyphenyl phosphate sodium salt produces a non-fluorescent product (resorcinol). The product then undergoes a nucleophilic reaction with dopamine to produce azamonardine, which is both chromogenic and fluorogenic (Scheme 4
). The assay was sensitive enough to give an LOD of 0.04 ng/mL and LDR of 0.125–8.0 ng/mL [35
The catalytic activity of certain nanoparticles was also utilized in enzyme-like activity for immunoassay. Miao et al. utilized the peroxidase-like properties of nanoceria to catalyze the oxidative conversion of o
-phenylenediamine (OPD) into 2,3-diaminophenazine (ox-OPD) (Scheme 5
) for dual colorimetric and ratiometric fluorescent signals detection modes. The effective fluorescence quenching of graphene QDs by the product results in a fluorescence spectral line with dual emission peaks in addition to a visible color change from colorless to orange [38
]. The assay had a low LOD (2.3 × 10−4
ng/mL) but with an LDR (0.001–10 ng/mL) lower than clinical concentrations of cTnI.
Another example of “artificial” enzymes apply the enzyme-like activity of [email protected]
nanodendrites. After capturing of cTnI and detection by [email protected]
nanodendrites-conjugated antibody, three methods are used to develop a signal that is directly dependent on cTnI concentration in solution. The first method, irradiation of the solution with a laser at 808 nm increases the temperature of the solution and it can be measured by a handheld thermometer. For the other two modes, the addition of OPD substrate to the solution in presence of H2
leads to its oxidation by [email protected]
nanodendrites to oxidized OPD (ox-OPD) (Scheme 5
) which can be detected by either a yellow color or an increase in fluorescence intensity at 580 nm upon introduction of highly fluorescent carbon dots [39
]. With a similar strategy, Tan et al. used Pd-Ir nanocubes to oxidize the nonfluorescent OPD to the fluorescent oxide product (oxOPD) in presence of H2
. The sandwiched immunoassay with this dual detection signal has an LOD of 0.31 pg/mL and an LDR of 0.001–1.0 ng/mL for cTnI [40