Overview on the Development of Alkaline-Phosphatase-Linked Optical Immunoassays

Abstract

The drive to achieve ultrasensitive target detection with exceptional efficiency and accuracy requires the advancement of immunoassays. Optical immunoassays have demonstrated significant potential in clinical diagnosis, food safety, environmental protection, and other fields. Through the innovative and feasible combination of enzyme catalysis and optical immunoassays, notable progress has been made in enhancing analytical performances. Among the kinds of reporter enzymes, alkaline phosphatase (ALP) stands out due to its high catalytic activity, elevated turnover number, and broad substrate specificity, rendering it an excellent candidate for the development of various immunoassays. This review provides a systematic evaluation of the advancements in optical immunoassays by employing ALP as the signal label, encompassing fluorescence, colorimetry, chemiluminescence, and surface-enhanced Raman scattering. Particular emphasis is placed on the fundamental signal amplification strategies employed in ALP-linked immunoassays. Furthermore, this work briefly discusses the proposed solutions and challenges that need to be addressed to further enhance the performances of ALP-linked immunoassays.

Keywords: alkaline phosphatase; chemiluminescence; colorimetry; fluorescence; immunoassays; surface-enhanced Raman scattering.

Figure 17

(A) Schematic illustration of the ALP-catalyzed dephosphorylation reaction for the sandwich plasmonic ELISA. (a) ALP catalyzes the dephosphorylation of ATP to generate adenosine and phosphate ions. (b) Dual-signal-amplified plasmonic ELISA based on the high loading of MBs and Zn²⁺-stimulated enzymatic reaction [143]. Copyright 2017 Elsevier. (B) Schematic illustration of naked-eye readout of plasmonic immunoassays based on ALP-triggered click chemistry [145]. Copyright 2014 American Chemical Society.

Figure 22

(A) Schematic illustration of AFP detection based on GTP-mediated enzyme cascade reaction [164]. Copyright 2018 Elsevier. (B) Schematic illustration of template-free just-in-time-producing copper hexacyanoferrate(III) strategy as oxidase-mimic-based colorimetric platform, enzyme-controllable-manner-based colorimetric platform, and CHNPs-ABTS-based colorimetric immunoassay [170]. Copyright 2022 Elsevier.

Scheme 1

Schematic diagram outlining the ALP-linked fluorescence immunoassays.

Figure 1

Schematic illustration of the microfluidic-laser-induced fluorescence immunosensor for determination of anti-T. gondii-specific antibodies [59]. Copyright 2019 Elsevier.

Figure 2

Schematic illustration of the fluorescence and colorimetric dual-mode immunoassay for detection of ZEN based on G4/NMM [64]. Copyright 2023 Elsevier.

Scheme 2

Chemical structures of ALP substrates and products as well as fluorogenic reactions.

Figure 3

(A) Schematic representation of fluorescence detection of AFP by coupling the conventional ELISA and ALP-guided fluorogenic reaction of AA and OPD [67]. Copyright 2019 American Chemical Society. (B) Schematic representation of the fluorescence detection of CEA by coupling AA2P/PTA reaction and the conventional ALP-based ELISA reaction [69]. Copyright 2021 American Chemical Society.

Figure 4

(A) Schematic illustration of turn-on fluorescence detection of IgG through the combination of the immunoreaction, ALP-mediated hydrolysis of BOIDPY-ATP, and Fe(III)-induced fluorescence quenching of BODIPY-ATP [72]. Copyright 2023 Elsevier. (B) Schematic representation of the fluorescence ELISA strategy via Pi-triggered fluorescence turn-on of the calcein−Ce³⁺complex [74]. Copyright 2018 American Chemical Society.

Figure 5

Schematic representation of the cascade ELISA strategy via tandem enzymatic fluorogenic and chromogenic reactions [75]. Copyright 2018 American Chemical Society.

Figure 6

Schematic illustration of (a) cascade reaction between CHzyme-catalyzed CAT oxidization and Schiff-base chemistry as well as catalytic substrate sensing mode for CAT. (b) Conventional oxidase-like CHzyme reaction used for biosensing. (c) Competitive sensing mode for AA. (d) Generated substrate sensing mode for ALP activity. (e) Application of substrate sensing mode for FELISA with the traditional ELISA in the contrast [79]. Copyright 2023 American Chemical Society.

Figure 7

(A) Schematic illustration of the IFE-based fluorescence immunosensor for detection of aflatoxin M₁ [86]. Copyright 2021 Elsevier. (B) Schematic illustration of the fluorescence immunoassay for detection of sulfamethazine based on GSH-capped AgNCs and ALP [90]. Copyright 2019 Elsevier.

Figure 8

Schematic illustration of the dual-mode fluorescent and colorimetric immunoassay for PSA detection based on in situ AA-induced signal generation from Fe-MOFs [96]. Copyright 2020 Elsevier.

Figure 9

(A) Schematic illustration of (a) process for the fabrication of CaCO₃–Au NPs/Ab₂/ALP bioconjugates and (b) immunosensor preparation on 96-well plates and the detection principle [97]. Copyright 2016 Elsevier. (B) Schematic illustration of the preparation procedure of Ab₂/AuNPs/DNA bioconjugates and immunoassay preparation on 96-well plates and sandwich-type detection procedure [98]. Copyright 2018 Elsevier.

Figure 10

(A) Schematic illustration of the fluorescence immunoassay for imidacloprid detection using ALP-catalyzed product to degrade the structure of CoOOH NSs [108]. Copyright 2019 Elsevier. (B) Schematic illustration of the fluorescence immunoassay for detection of amantadine using the nanoassembly of CDs and MnO₂ NSs as the AA-responsive signal probe [110]. Copyright 2019 Elsevier.

Figure 11

(A) Schematic illustration of fluorescence ELISA strategy through ALP-triggered in situ synthesis of dsDNA-templated CuNCs [111]. Copyright 2019 Elsevier. (B) Schematic illustration of the fluorescent ELISA strategy via ALP-enabled in situ synthesis of SiNPs [114]. Copyright 2016 American Chemical Society.

Figure 12

Schematic illustration of detection of anti-BSA antibody based on enzymatic growth of CdS QDs [115]. Copyright 2013 American Chemical Society.

Figure 13

(A) Schematic illustration of the AIE immunosensor for IgG detection based on PPi-triggered TPDA aggregation [120]. Copyright 2019 Elsevier. (B) Schematic illustration of the Ce⁴⁺/Ce³⁺-triggered dual-readout immunoassay for OTA detection based on AIE effect and the oxidation of TMB [122]. Copyright 2022 Elsevier.

Scheme 3

Schematic diagram outlining the ALP-linked colorimetric immunoassays.

Scheme 4

Chemical structures of ALP substrates and products as well as chromogenic reactions.

Figure 14

(A) Schematic illustration of working principle of the ALP-catalytic color development system with Cu(II)-BCA as a chromogen [133]. Copyright 2018 Elsevier. (B) Schematic illustration of the cascade-amplified colorimetric immunoassay using ALP/anti-CEA@ZnCPs as a detection antibody [134]. Copyright 2019 American Chemical Society.

Figure 15

Schematic illustration of the chemical-redox-cycling-based colorimetric ELISA for AFP detection [135]. Copyright 2019 American Chemical Society.

Figure 16

Schematic illustration of ALP-triggered ratiometric colorimetric and fluorescence immunosensor based on NA-ALP fusion protein for detection of fenitrothion [137]. Copyright 2022 Elsevier.

Figure 18

Schematic illustration of peptide-ALP-AuNPs immunoassay for simultaneous detection of multiple inflammatory markers:interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) [146]. Copyright 2018 American Chemical Society.

Figure 19

Schematic illustration of the enzyme-induced metallization-based colorimetric assay [148]. Copyright 2014 American Chemical Society.

Figure 20

(A) Schematic illustration of the principle for multicolor visual detection of HER2 ECD based on NADH-assisted AA-mediated growth of AuNBPs together with antibody [151]. Copyright 2019 American Chemical Society. (B) Schematic illustration of the competitive colorimetric ELISA for detection of XAA based on the multicolor change of AuNRs [152]. Copyright 2022 American Chemical Society. (C) Schematic illustration of the plasmonic and photothermal immunoassay through ALP-triggered crystal growth on AuNSs [153]. Copyright 2019 American Chemical Society. (D) Schematic illustration of the plasmonic immunoassay for detection of IL-1β with ALP-triggered geometrical transformation of AgNPLs [154]. Copyright 2023 American Chemical Society.

Figure 21

Schematic illustration of visual plasmonic ELISA for IgG detection based on ALP-triggered etching of AuNRs [162]. Copyright 2015 American Chemical Society.

Figure 23

Schematic illustration of the colorimetric immunoassay for detection of mouse IgG based on enzymatic cascade reaction by combining ALP catalysis and in situ generation of photoresponsive nanozyme TiO₂-CA [171]. Copyright 2015 American Chemical Society.

Scheme 5

Chemical structures of ALP substrates and products as well as chemiluminescence reactions.

Figure 24

(A) Schematic illustration of the preparation of the ALP and HRP-based nanosensors and simultaneous detection of AFP and Golgi protein 73 [184]. Copyright 2021 Elsevier. (B) Schematic illustration of an ALP/luciferase double-enzyme-mediated bioluminescent biosensor for POCT of PCT [185]. Copyright 2017 American Chemical Society.

Figure 25

Schematic illustration of DNA-nanofirecracker-based ultrasensitive SERS immunoassay for tPSA detection [197]. Copyright 2020 American Chemical Society.