Electrochemiluminescent (ECL) biosensor for Burkholderia pseudomallei based on cobalt-doped MOF decorated with gold nanoparticles and N-(4-aminobutyl)-N-(ethylisoluminol).

A multifunctional catalytic nanomaterial (Co-MOF@AuNP@ABEI) composed of cobalt-doped metal-organic frameworks (Co-MOF), gold nanoparticles (AuNP), and N-(4-aminobutyl)-N-(ethylisoluminol) (ABEI) is reported. Co-MOF@AuNP@ABEI exhibits high synergistic and zero-distance catalytic properties, which are beneficial to the improvement of the detection sensitivity of an electrochemiluminescent (ECL) biosensor. After coupling with the ECL system and 3D magnetic walking nanomachine amplification strategy, the Co-MOF@AuNP@ABEI can achieve an ultrasensitive ECL assay of Burkholderia pseudomallei with the limit of detection (LOD) of 60.3 aM, which is 2 and 4 orders of magnitude lower than individual ECL system without the nanomachine (4.97 fM) and individual walking nanomachine (340 fM), and superior to the pathogenic bacteria analyses in the previous report. Moreover, the LOD of the proposed ECL detection system for the determination of B. pseudomallei in serum sample was as low as 9.0 CFU mL-1. The relative standard deviations (RSD) of ECL intensity for the detection of five B. pseudomallei-spiked serum samples were 4.02%, 0.84%, 0.84%, 1.55%, and 0.21%, respectively. The recoveries of the ECL biosensor for the detection of B. pseudomallei DNA-spiked serum samples were 93.63 ~ 107.83%. Therefore, this work demonstrated that the developed multifunctional catalytic nanomaterial with synergistic and zero-distance catalytic properties can be used as excellent ECL signal reporter to improve the detection sensitivity of ECL biosensor.

An electrochemiluminescence immunosensor based on ABEI-GO-AgNPs as a double-amplified luminophore for the ultra-sensitive detection of prostate-specific antigen.

A sandwich electrochemiluminescence (ECL) immunosensor based on an N-(4-aminobutyl)-N-ethylisoluminol-graphene oxide-Ag nanoparticle (ABEI-GO-AgNPs) complex and cysteine silver nanowires (AgCysNWs) was prepared to detect prostate-specific antigen (PSA). Our results showed that an ECL signal probe, ABEI-GO-AgNPs, with an ultrahigh specific surface area, favorable catalytic properties, and electrical conductivity was prepared by a one-step synthesis method. ABEI-GO-AgNPs with good biocompatibility immobilized secondary antibody (Ab2) via AgN bonds. Furthermore, AgCysNWs containing many -COOH groups were prepared and used to enrich primary antibody (Ab1), which could be used as an affinity probe for the selective capture of PSA. Lastly, through layer-by-layer assembly, we established an ECL immunosensing platform for the sensitive detection of PSA. Under the optimized conditions, the designed ECL immunosensor showed promising sensitivity and selectivity for the detection of PSA in the linear range of 5.5 × 10-7-5.5 ng/mL, with a detection limit of 1.2 × 10-7 ng/mL. The constructed ECL sensing platform possessed good specificity, reproducibility, and stability and could detect PSA in actual human serum samples.

Ultrasensitive electrochemiluminescence aptasensor based on ABEI reduced silver nanoparticles for the detection of profenofos.

An ultrasensitive electrochemiluminescence (ECL) aptasensor for detection of profenofos was constructed by the reducibility and chemiluminescence property of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). ABEI was used to reduce silver nitrate (AgNO3) to silver nanoparticles (AgNPs), which could be adsorbed on the lattice of graphene oxide (GO) to form ABEI-AgNPs-GO complex. This compound could achieve excellent luminescence. The aptamer (Apt) modified (5') by sulfhydryl groups could be immobilized on AgNPs to capture profenofos. When profenofos was present, the ECL signal of the aptasensor would be weakened. To further demonstrate the successful construction of the aptasensor, cyclic voltammetry tests were performed on an electrochemical workstation and an ECL analyzer, respectively. The standard curve and specificity experiment both showed that the sensor had the advantages of low limit of detection (LOD) and good specificity. Under the optimal conditions, the aptasensor had a good linear response for profenofos in the range of 1 × 10-1-1 × 104 ng/mL. It also had a LOD of 6.7 × 10-2 ng/mL and a correlation coefficient (R2) of 0.9991. The aptasensor had been successfully applied to the detection of profenofos in vegetables. The recovery range of the proposed ECL aptasensor was 98 % ~ 107.4 %.

Efficient ABEI-Dissolved O2-Ce(III, IV)-MOF Ternary Electrochemiluminescent System Combined with Self-Assembled Microfluidic Chips for Bioanalysis.

A signal-amplified electrochemiluminescent (ECL) sensor chip was developed for sensitive analysis of procalcitonin (PCT). Herein, we first prepared a self-enhanced luminophore, which enhanced ECL responses through intramolecular reactions. Second, Au-Pd bimetallic nanocrystals and mixed-valence Ce-based metal-organic frameworks (MOFs) were introduced as co-reaction promoters to facilitate the reduction of dissolved O2. Based on the synergistic catalysis of Au and Pd, the spontaneous cyclic reaction of Ce(III)/Ce(IV), and the high electrochemical active surface area of Ce(III, IV) MOF, a large number of superoxide anion radicals (O2•-) and hydroxyl radicals (OH•) were produced. Therefore, the luminescence efficiency of N-(aminobutyl)-N-(ethylisoluminol)-dissolved O2 (ABEI-O2) systems were greatly improved, providing a new prospect for the application of dissolved O2 in ECL analysis. In addition, the affinity peptide ligands were used for the directional connection of antibodies to provide protection for the bioactivity of the proposed sensor. Finally, the microfluidic technology was applied to ECL analysis to integrate the three-electrode detection system into the self-assembled microfluidic chip, which realized the automation and portability of the detection process. The developed sensor showed high sensitivity for PCT detection with a detection limit of 3.46 fg/mL, which possessed positive significance for the clinical diagnosis of sepsis.

Highly sensitive competitive electrochemiluminescence immunosensor based on ABEI-H2O2 system with cobalt hydroxide nanosheets and bimetal PdAg as co-enhancer for detection of florfenicol.

A competitive electrochemiluminescence immunoassay was established based on the isoluminol-H2O2 (ABEI-H2O2) system catalyzed by cobalt hydroxide (Co(OH)2) to detect florfenicol residues in food. First , ultra-thin two-dimensional Co(OH)2 nanosheets were used as the catalyst of ABEI-H2O2 system, and excellent catalytic effects were acquired by catalytic decomposition of hydrogen peroxide with cobalt ions. Then, bimetal PdAg (Pd/Ag) alloy nanoparticles were used as a bridge to connect ABEI and antibody due to their good biocompatibility; Pd/Ag alloy nanoparticles also had a catalytic effect to further amplify the ECL signal in the system due to the synergistic catalytic effect of the bimetal. A competitive immunoassay strategy was used to detect florfenicol, where the florfenicol in the sample will compete with the antibody for the limited binding sites on the coating antigen. The ECL immunosensor for florfenicol detection shows high sensitivity, with a linear range from 10-4  to 102 ng mL-1, and a detection limit of 3.1 × 10-5 ng mL-1, where the scan potential was varied from 0 to 0.6 V vs Ag/AgCl . This work was the first to use Co(OH)2 nanosheets and bimetal PdAg catalytic signal amplification methods to design the sensor, which provides a novel, convenient and reliable strategy for ultra-sensitive detection of florfenicol, and other biological small molecules. A novel ECL immunosensor based on ABEI-H2O22.

Potent 2,3-dihydrophthalazine-1,4-dione derivatives as dual inhibitors for mono-ADP-ribosyltransferases PARP10 and PARP15.

While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130-160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.

MicroRNA-21 electrochemiluminescence biosensor based on Co-MOF-N-(4-aminobutyl)-N-ethylisoluminol/Ti3C2Tx composite and duplex-specific nuclease-assisted signal amplification.

A novel electrochemiluminescence (ECL) biosensor for the determination of microRNA-21 (miRNA-21) was developed, based on a hybrid luminescent Co-MOF-ABEI/Ti3C2Tx composite as an ECL luminophore combined with a duplex-specific nuclease (DSN)-assisted signal amplification strategy. The synthesized Co-MOF-ABEI/Ti3C2Tx composite carrying N-(4-aminobutyl)-N-ethylisoluminol (ABEI) exhibited strong and stable ECL in the presence of reactive oxygen species (ROS). The ECL biosensor was fabricated by adsorbing Co-MOF-ABEI/Ti3C2Tx onto a glassy carbon electrode and covalently coupling the probe DNA onto the surface of the Co-MOF-ABEI/Ti3C2Tx-modified electrode. In the presence of the target miRNA-21, the DSN selectively cleaved the complementary DNA section (S1) to miRNA-21, resulting in the release of the transduction section (S2) and the reuse of miRNA-21 in the subsequent amplification cycle. The interaction of the stem-loop structure of the probe DNA with the Co-MOF-ABEI/Ti3C2Tx-modified glassy carbon electrode with S2 strands led to the opening of the annular part of the probe DNA. Then, the opened guanine (G)-rich sequences of probe DNA were exposed and folded into a hemin/G-quadruplex DNAzyme in the presence of hemin. The catalysis of H2O2 to ROS by the hemin/G-quadruplex DNAzyme significantly enhanced ECL intensity, and this intensity was logarithmically proportional to the concentration of target miRNA-21 between 0.00001 and 10 nM, having a limit of detection of 3.7 fM. The designed ECL biosensor can  detect miRNA-21 extracted from HeLa cells, indicating its promising application in clinical diagnosis and disease prognosis analysis.

Performances of a novel chemiluninescence ABEI-based NT-proBNP immunoassay.

Natriuretic peptides are widely used in clinical practice as cardiac markers for early diagnosis, prognosis and for the monitoring of treatment efficiency of heart Failure (HF). According to the clinical relevance of natriuretic peptides testing, it is important to assess the performances of novel platform for testing. Our study showed the overall good performances of a new NT-proBNP ABEI-based automated immunoassay.

Electrochemiluminescence from a biocatalysis accelerated N-(aminobutyl)-N-(ethylisoluminol)/dissolved O2 system for microRNA detection.

A kind of biocatalyst, laccase, has been employed as a biocompatible coreactant accelerator to efficiently catalyze coreactant (dissolved O2) for generating high local concentration of superoxide radical (O2•-), acquiring high-intense electrochemiluminescence (ECL) emission of ABEI (N-(aminobutyl)-N-(ethylisoluminol))/dissolved O2 system. Furthermore, a modified strand displacement reaction with excellent amplification efficiency was constructed by replacing traditional single strand DNA to the hairpin DNA as template for triggering the immobilization of more signal probes. As a result, the biosensor for microRNA-21 determination has preeminent selectivity and favorable sensitivity with detection limit down to 80.8 aM. Significantly, the devised strategy has blazed a new path for seeking more coreaction accelerators with splendid biocompatibility thus promoting the application of ternary ECL systems in biological analysis and clinical diagnosis.

Novel cobalt-based metal-organic frameworks with superior catalytic performance on N-(4-aminobutyl)-N-ethylisoluminol chemiluminescent reaction.

Novel cobalt-based metal-organic frameworks (Co MOFs) were synthesized by a facile "controlled synthesis" strategy. The MOFs displayed superior catalytic performance on the chemiluminescent (CL) reaction between N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and H2O2. UV-vis absorption, CL spectrum, ESR, and radical scavenger experiments were conducted for clarifying the catalytic mechanism of Co MOFs. All results revealed that Co MOFs can accelerate decomposition of H2O2 and production of OH•, O2•-as well as 1O2 radicals. The rapid reaction between these reactive oxygen species and ABEI resulted in the generation of ABEI-ox∗. The excited-state oxidation product emitted a very intensive CL signal with a maximal emission wavelength of 430 nm as it returned to the ground state. To explore their application potential in CL assay, Co MOFs were used as powerful CL reaction catalyst for establishing a very sensitive method for immunoassay of aflatoxin B1. The detection range was 0.05-60 ng mL-1, and the limit of detection was 4.3 pg mL-1. The result for detecting herbal medicine samples demonstrates the acceptable reliability of the Co MOFs-based CL immunoassay. The proof-of-principle work verifies the application potential of Co MOFs on boosting intensive CL signal, and meets the demand for high sensitivity in various bioassay fields.

BODIPY and 2,3-Dihydrophthalazine-1,4-Dione Conjugates As Heavy Atom-Free Chemiluminogenic Photosensitizers.

We disclose an interesting concept for developing heavy atom-free chemiluminogenic photosensitizers. To accomplish this, conjugates 2 and 3, which are composed of boron-dipyrromethene (BODIPY) and 2,3-dihydrophthalazine-1,4-dione units, are investigated. 2 and 3 are compared in terms of their photophysical properties, chemiluminescence responses, and singlet oxygen production. Strikingly, the results indicate that decoration of BODIPY with the 2,3-dihydrophthalazine-1,4-dione scaffold boosts the singlet oxygen generation. Furthermore, treatment of epidermoid laryngeal carcinoma Hep-2 (Hep-2) cells with conjugates 2 and 3 results in efficient cellular internalization which ensures live- cell imaging of Hep-2 cells. Finally, it is noteworthy that in vitro cytotoxicity assays reveal that both 2 and 3 induce cytotoxicity when illuminated with red light. Thus, 2 and 3 represent heavy atom-free chemiluminogenic photosensitizers.

Immune-Modulating Drug MP1032 with SARS-CoV-2 Antiviral Activity In Vitro: A potential Multi-Target Approach for Prevention and Early Intervention Treatment of COVID-19.

At least since March 2020, the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic and the multi-organ coronavirus disease 2019 (COVID-19) are keeping a firm grip on the world. Although most cases are mild, older patients and those with co-morbidities are at increased risk of developing a cytokine storm, characterized by a systemic inflammatory response leading to acute respiratory distress syndrome and organ failure. The present paper focuses on the small molecule MP1032, describes its mode of action, and gives rationale why it is a promising option for the prevention/treatment of the SARS-CoV-2-induced cytokine storm. MP1032 is a phase-pure anhydrous polymorph of 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt that exhibits good stability and bioavailability. The physiological action of MP1032 is based on a multi-target mechanism including localized, self-limiting reactive oxygen species (ROS) scavenging activities that were demonstrated in a model of lipopolysaccharide (LPS)-induced joint inflammation. Furthermore, its immune-regulatory and PARP-1-modulating properties, coupled with antiviral effects against SARS-CoV-2, have been demonstrated in various cell models. Preclinical efficacy was elucidated in LPS-induced endotoxemia, a model with heightened innate immune responses that shares many similarities to COVID-19. So far, during oral clinical development with three-month daily administrations, no serious adverse drug reactions occurred, highlighting the outstanding safety profile of MP1032.

Novel Protease-Free Long-Lasting Chemiluminescence System Based on the Dox-ABEI Chimeric Magnetic DNA Hydrogel for Ultrasensitive Immunoassay.

Most of chemiluminescence (CL) substrates exhibit the flash-type light emission. Therefore, the long-lasting CL system is always the crown in the field of CL-based analysis methodology. In this work, we constructed a Dox-ABEI chimeric magnetic DNA hydrogel (MDH) as a novel protease-free long-lasting CL reaction system. The functional MDH can transform flash-type ABEI/H2O2/CO2+ reaction into a glow-type CL system because of its block effect on delaying the diffusion rate of co-reactants, making the CL reaction gradually occur. More importantly, the functional MDH possessed the advantages of biocompatibility and controllability and could be well-designed to incorporate different biosensing strategies. Subsequently, we established a functional MDH-based long-lasting CL immunoassay system for ultrasensitive and highly specific detection of d-dimer and fibrin degradation products (FDPs). The designed CL immunoassay can detect d-dimer and FDP down to 53.7 and 31.6 fg/mL, respectively, with a wide line ranging from 100 fg/mL to 100 ng/mL, which was superior to the previously reported CL biosensing strategies. Moreover, benefiting from the magnetic separation of MDH and excellent CL performance, the developed immunoassaying method was successfully applied in the detection of clinical samples, which showed a close correlation with clinical reference technology. Thus, this functional MDH proved to be an excellent long-lasting CL system and a potential technical platform for clinical bioanalysis applications.

Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex DNAzyme Nanostructures.

Catalytic nanostructures have the potency to mimic enzymatic features. In this paper, we show that the complex between hemin and G-quadruplex DNA efficiently catalyzes the modification of proteins with N-methyl luminol derivatives. Final conversions are reached within 15-30 min, and LC-MS analysis of tryptic digests of the proteins shows that the reaction proceeds with chemoselectivity for electron-rich aromatic residues (Tyr ≫ Trp), and the site-specificity of the modification depends on the sequence and secondary structure folding of the G-quadruplex nanostructure. Furthermore, the modification can be applied on proteins with different biomedical functions, and the nanostructure can be designed to contain a regulatory element in order to regulate protein modification by an external stimulus.

Simple and selective optical biosensor using Ultrasonicator synthesis of 5-((anthracen-9-ylmethylene) amino)-2,3-dihydrophthalazine-1,4-dione for direct detection of ascorbic acid in vegetables and fruits.

We report an optical biosensor using imine, 5-((anthrcene-9-ylmethylene) amino)-2,3dihydrophthalazine) 1-4-dione (ADD) for direct detection of ascorbic acid (AA) via FRET quenched. The ADD was successfully prepared by using simple ultra - sonication method, which was characterized by various spectroscopic techniques. The fluorescence intensity of ADD probe was drastically quenched in presence of AA, and shown excellent selectivity towards the detection of AA in presence of possible biological active interferences. A wide linear range from 0.25 to 190 µM was achieved towards the detection of AA with a LOD of 10 nM. The occurrence of FRET mechanism is due to intermolecular hydrogen bonding between ADD and AA, which was confirmed by Density Functional Theory calculations. Moreover, the biosensor was successfully applied for the detection of AA in real samples such as fruits and vegetables to demonstrate the practicability. In addition, the developed biosensor could be a simple and economically cheap platform for the detection of AA in food samples.

Metal Ion-Mediated Potential-Resolved Ratiometric Electrochemiluminescence Bioassay for Efficient Determination of miR-133a in Early Diagnosis of Acute Myocardial Infarction.

Potential-resolved electrochemiluminescence (PRECL)-based strategies utilizing novel nanoluminophores have become a research hotspot in analytical sciences in virtue of the superior versatility, specificity, and sensitivity they offer to bioassays. In this work, novel PRECL nanoluminophore graphitic carbon nitrides (g-C3N4) functionalized by N-(aminobutyl)-N-(ethylisoluminol) (ABEI) (g-C3N4/ABEI) were developed, exhibiting two potential-resolved ECL emissions in the presence of H2O2/K2S2O8 as coreactants with the peak potentials at +1.2 and -1.6 V, respectively. It was found that Hg2+ could simultaneously decrease the ABEI electrochemiluminescence (ECL) intensity while increasing the intensity of g-C3N4 on the contrary. Using Hg2+ as the ECL signal moderator based on specific interactions between Hg2+ and thymine-thymine pairs, conjoined with the synthesized g-C3N4/ABEI/Hg2+ nanohybrids with outstanding PRECL properties as the sensing interface, a label-free and all-in-one ratiometric ECL bioassay combined with a rolling circle amplification (RCA) strategy was designed for the detection of miR-133a as a biomarker in early diagnosis of acute myocardial infarction (AMI). The proposed bioassay provided a quantitative readout proportional to the target miR-133a concentrations in the range from 0.1 fM to 1.0 pM with a detection limit of 48.0 aM. Owing to its inherent merits for an effective amplification of the ECL signals ratio and a simple one-step assembly procedure, the proposed bioassay demonstrated excellent analytical performance with remarkable sensitivity, specificity, and low measurement deviations, manifesting its potential application in early AMI diagnosis.

Dual-modality probe based on black phosphorous and NiFe2O4 NTs for electrochemiluminescence and photothermal detection of ovarian cancer marker.

An electrochemiluminescence and photothermal immunosensor based on a dual-modality integrated probe was proposed for sensitive and reliable detection of lipolysis stimulated lipoprotein receptor (LSR), a new biomarker of ovarian cancer. Black phosphorous quantum dots (BPQDs) possess fascinating electrochemical property and unique photothermal effect, which could not only enhance ECL signal of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) through accelerating dissolved O2 evolution but also realize temperature signal output by converting laser energy into heat. Furthermore, NiFe2O4 nanotubes (NiFe2O4 NTs) have large specific surface area and favorable adsorption ability, which could increase the immobilized amount of ABEI and BPQDs, further strengthening ECL and temperature signal. As a result, a dual-mode immunosensor was constructed and realized ECL and temperature dual signal to detect LSR, making the results more reliable. This work provided a new thought for the development of sensitive and accurate sensors and was expected to employ for determination of other biomarkers.

Cu(II)-Regulated On-Site Assembly of Highly Chemiluminescent Multifunctionalized Carbon Nanotubes for Inorganic Pyrophosphatase Activity Determination.

A novel signal-on chemiluminescence (CL) assay for pyrophosphatase (PPase) activity determination was innovatively developed based on the Cu(II)-regulated on-site assembly of highly chemiluminescent Cu(II), N-(aminobutyl)-N-(ethylisoluminol) (ABEI), gold nanodot, and chitosan multifunctionalized carbon nanotubes (Cu(II)/ABEI-Au/cs-CNTs). First, ABEI-functionalized gold nanodots (ABEI-Au) were assembled on the surface of chitosan-modified carbon nanotubes (cs-CNTs) via the reduction of HAuCl4 with ABEI in a cs-CNT suspension to form ABEI-Au/cs-CNTs. Then, it was found that the catalyst Cu(II) can be selectively, efficiently, and quickly adsorbed onto ABEI-Au/cs-CNTs via the high-affinity interactions between Cu(II) and cs-CNTs to form novel hybrid nanomaterials Cu(II)/ABEI-Au/cs-CNTs. The CL intensity of Cu(II)/ABEI-Au/cs-CNTs was enhanced by about 2 orders of magnitude compared with that of ABEI-Au/cs-CNTs. Furthermore, it was found that in the presence of pyrophosphate ions (PPi), PPi could coordinate with Cu(II) to form a stable PPi-Cu(II) complex and block the assembly of Cu(II)/ABEI-Au/cs-CNTs. After the addition of PPase, PPase could catalyze the hydrolysis of PPi into Pi and release Cu(II) from the PPi-Cu(II) complex. The released free Cu(II) could trigger the on-site assembly of highly chemiluminescent Cu(II)/ABEI-Au/cs-CNTs, resulting in an enhanced CL intensity. The enhanced CL intensity had a good linear relationship with the activity units of PPase ranging from 0.025 to 0.5 U, with a detection limit of 9 mU. The method was employed to monitor the PPase inhibitor efficiently. Cu(II)/ABEI-Au/cs-CNTs with excellent CL may also find more applications in the development of novel CL analytical methods.

[Development and Application of Catalytic Tyrosine Chemical Modification].

The chemical labeling of proteins with synthetic small compounds is a key technique in chemical biology, protein-based therapy, and material science. Much of the chemical labeling of native proteins, however, depends on the labeling of lysine and cysteine residues. While those methods have contributed significantly to native protein labeling, alternative methods that can modify different amino acid residues are still required. Here we report the development of a novel methodology of oxidative tyrosine labeling, which was inspired by the single-electron transfer reaction in biological systems. The tyrosine labeling methods were developed using small compounds such as N-methyl luminol derivative, N'-acyl-N,N-phenylenediamine, and 1-methyl-4-aryl-urazole under labeling conditions using a hemin, peroxidase, or ruthenium photocatalyst. These methods were applied to target- and site-selective protein modification.

A ratiometric electrochemiluminescent immunoassay for calcitonin by using N-(aminobutyl)-N-(ethylisoluminol) and graphite-like carbon nitride.

A ratiometric electrochemiluminescent (ECL) assay is described for the determination of the calcium(II) regulator calcitonin (CT). The method is making use of (a) graphite-like carbon nitride (g-C3N4) as the cathodic luminophore, (b) N-(aminobutyl)-N-(ethylisoluminol) (ABEI) as the anodic luminophore, and (c) peroxodisulfate and dissolved oxygen as coreactants. The luminous potential of g-C3N4 and ABEI can be well distinguished because of their different luminescent properties. Energy transfer between g-C3N4 and ABEI is not observed, and the coreactants peroxodisulate and oxygen do not interfere with each other. Au nanoparticles were functionalized with g-C3N4 and placed on the electrode to serve as a matrix for immobilization of primary antibody (Ab1). In the presence of CT, it will bind to the electrode. Then secondary antibody (Ab2) modified with polyaniline (PANI) and ABEI is incubated onto the electrode. With the increase in the concentration of CT, the blue ECL of g-C3N4 is quenched by PANI, while the blue luminescence of ABEI is enhanced. This enables ratiometric detection of calcitonin by ratioing the internsities at 460 and 475 nm. Response is linear in the 0.1~40 pg·mL-1 CT concentration range, and the limit of detection is 23 fg·mL-1. The method breaks the limitation of common ECL ratiometric strategy, namely, two luminophores often share the common coreactant. Graphical abstractSchematic representation of an immunoassay where polyaniline (PANI) in a BSA-Ab2-ABEI-Au@PANI composite quenches the cathodic signal of a graphitic carbon nitride (Au-g-C3N4) modified with gold nanoparticles (Au), while N-(aminobutyl)-N-(ethylisolumino) (ABEI) in the BSA-Ab2-ABEI-Au@PANI composit produces an anodic signal that enables quantitation of calcitonin.