The amperometric HbA1c sensor detects biomolecules by the change in current as the output signal. This type of sensor was first developed in 2002, in which HbA1c molecules were attached to the electrode surface by a cellulose membrane pre-modified with globin [29]. This ground-breaking work verified the capability and promising potential of amperometric sensors for detecting HbA1c. Moreover, antibodies, boric acid and its derivatives, ferrocene and its derivatives, and nucleic acid aptamers can be used to construct amperometric sensors [22,25,30]. Since HbA1c is a protein with reduction property, in general, the detection mechanism of the amperometric HbA1c sensor is that the electrode modification substance oxidizes HbA1c to produce a redox reaction, or HbA1c hinders the oxidation current value of redox media [31,32].

Boric acid-based. Under weakly alkaline conditions, boric acid can covalently bind to the diastatic cis-diol bonds on the surface of HbA1c [33]. Song et al. [34] proposed a method of using boric acid-polyamine G4 dendrimer-modified electrodes, verified successful binding of HbA1c at a content ranging from 2.5% to 15%, and simultaneously measured the electrochemical current generated as ferrocene methanol was catalyzed by glucose oxidase (GO_x) on the electrode surface. The current value can be used as an indicator of the combination of HbA1c and the boric acid layer. Furthermore, to detect HbA1c in whole human blood, Song et al. developed a competitive electrochemical HbA1c biosensor based on the cis-diol interaction between HbA1c and a boronate recognition group [35]. Predetermined concentrations of HbA1c and activated GO_x were simultaneously dropped onto the surface of boric acid-modified electrodes, and the two species competed for the limited binding sites. The experimental results provided a linear response within the content range of 4.5–15%. This biosensor holds great potential for the determination of HbA1c in whole blood samples without labeling with antibodies, dyes, or fluorescent materials. However, since boric acid can also combine with other sugar substances [36], the detection specificity is poor, and the entire blood sample needs to be pretreated before detection. Recently, a certain modification of boric acid was performed to achieve specific binding of HbA1c. Thiruppathi et al. developed a dual-electrode sensor (SPCE/CNT-Nf@Hb-Nf and SPCE*/AQBA-HbA1c) to detect Hb and HbA1c in whole blood samples simultaneously. Anthraquinone boric acid was prepared through electrooxidation with anthracene boric acid as the raw material. The SPCE*/AQBA electrode could be identified using a specific borate-diol and recombined with the applied HbA1c [18].

Phenylboronic acid (PBA) is obtained by replacing one of the hydroxyl groups in boric acid with a phenyl group. This compound can bind HbA1c by a borate bond, and the catalyst or redox-active species should be attached to the electrode to obtain an electrical signal [37]. A poly(terthiophene benzoic acid) (pTTBA)-modified electrode was immersed in the HbA1c solution, and the current generated from the reduction reaction between HbA1c and hydrogen peroxide (H₂O₂) was used to determine the HbA1c level. The linear dynamic detection range varied only from 0.1 to 1.5% [38], and it was not suitable for clinical use. Another research group, Chopra et al., used a conducting self-assembled monolayer (SAM) of mercaptophenyl boronic acid (MPBA) to bind HbA1c [39]. The authors labelled a gold screen-printed electrode with a ferrocene-tagged anti-HbA1c antibody (FcAb) as a tracer molecule. The produced current was proportional to the amount of HbA1c between 5% and 16%, significantly expanding the detection range. In another study, researchers coated a layer of phenylboronic acid-modified pyrroloquinoline quinine (PBA-PQQ) onto the surface of a glassy carbon disc electrode [40] (Figure 2C). The HbA1c captured on the electrode surface led to the reduction in the oxidation peak current of PQQ, because the protein molecules hinder the electron transfer pathway. Within the HbA1c concentration range of 9.4–65.8 µg/mL, the peak current decreased linearly by differential pulse voltammetry (DPV). Similarly, carbon electrodes coated with polyaniline boric acid nanoparticles were also used to determine HbA1c in a label-free manner [41]. The peak current varied according to a linear relationship with the logarithm of HbA1c concentration within the range of 0.975–156 µM, with high selectivity.

In recent years, some novel electrochemical HbA1c sensors have been reported. A reticulated vitreous carbon (RVC) electrode was modified with 3-aminophenylboronic acid, chitosan (CHIT), and tetraethyl silica (TEOS) [32]. The biosensor was employed to detect HbA1c in clinical samples, and comparison showed that the detection results were basically the same as those from automatic biochemical analysis. In 2018, a molecularly imprinted polymer (MIP) flexible sensor was reported to simultaneously detect HbA1c and Hb by specifically capturing targets through non-covalent bonding and a cis-diol structure [43]. The linear ranges for detecting HbA1c and Hb were 0.2–230 ng/mL and 0.5–200 ng/mL, respectively. The sensor was successfully applied to determine the concentration of HbA1c in blood samples collected from women with gestational diabetes and healthy pregnant women. A novel graphene-doped titanium dioxide (TiO₂)-based heterojunction nano hybrid material (HJNH) was modified by poly(3-aminophenylboric acid) (PAPBA) and gold nanoparticles (AuNPs) [44]. The boric acid group in PAPBA was used to capture HbA1c. When the content of HbA1c ranged from 2.0% to 10%, the signal was directly proportional to the electrocatalytic reduction current of H₂O₂, and the detection limit was 0.17%. In 2019, another type of unlabeled electrochemical sensor was developed for HbA1c detection. In this sensor, 4-mercaptophenyl boric acid (4-MPBA)-modified screen-printed electrodes modified by gold nanoflowers (AuNFs) were used as the sensing electrodes [42]. The linear range for detecting HbA1c was 5–1000 µg/mL or 2–20% of the content, and the results demonstrated that the sensor had good specificity and stability. In addition, the use of a 16-channel screen-printed electrode and the considerably reduced detection time and cost endowed this sensor with significant performance superiority over other existing HbA1c detection methods in terms of pretreatment and operation procedures. Recently, to improve the electrode surface electron transmission capacity, Li et al. designed a three-dimensional antifouling nano-biosensing surface based on bovine serum albumin (BSA) and glutaraldehyde (GA) cross-linking and then used the HbA1c antibody and 3-aminophenyl boronic acid (APBA) to functionally modify the surface [45]. The presence of non-glycated hemoglobin (HbAo) resulted in a linear dynamic range of 2–15%, which facilitated label-free POCT detection of HbA1c. A redox medium was fixed on the surface of a nanocomposite, which contained pTTBA and N,S-doped porous carbon (NSPC), to fabricate HbA1c sensors [46]. This system could accurately separate and detect Hb and HbA1c in blood samples. The linear dynamic ranges of Hb and HbA1c were 1.0 × 10⁻⁶–3.5 mM and 3.0 × 10⁻⁶–0.6 mM, respectively.

Ferrocene-based. Ferrocene (Fc)-modified electrodes may be promising for the construction of current sensors because of the reasonable stability and structural versatility of Fc derivatives [23,47,48]. In a recent study, Han et al. reported a novel scheme that made full use of the redox ability of ferrocene diformylcysteine (Fc[CO-Cys(Trt)-OMe]₂) and ferrocene glutathione (Fc[CO-Glu-Cys-Gly-OH]) [49]. The two derivatives were adsorbed on the surface of electrodes modified by AuNPs, and the performance of the sensor was quantitatively characterized. The ferrocene glutathione sensor was proven to have a stronger catalytic current response to Hb, and the current showed a good linear correlation, with Hb concentrations ranging from 0.1 to 1000 µg/mL. The relative standard deviation was less than 4.7%, and the recovery rate was between 95.5% and 103.2%. Both properties meet the clinical requirements for Hb analysis.

Aptamer-based. A nucleic acid aptamer is a sequence of short single-strand DNA or RNA, and aptamers are produced through in vitro selection procedures. Aptamers provide several unique advantages: minimal possibility of chemical synthesis, small batch variability, long shelf life, stability under various conditions, and a variety of available chemical modifications. Therefore, aptamers are now the most promising alternative to monoclonal antibodies [50]. Novel aptamers for glycated and total hemoglobin have been selected recently, showing high affinity and specificity [51].

Kim et al. reported a dual sensor for detecting HbA1c and Hb in a finger prick blood sample (1 µL) [17]. The Hb content was determined by measuring the cathode current generated from catalysis with toluidine blue O (TBO), while the HbA1c content was determined by measuring the cathode current produced when HbA1c was captured by the aptamer. The dynamic ranges for detecting Hb and HbA1c were 0.1–10 µM and 0.006–0.74 µM, respectively, and the mean HbA1c values (%) of the proposed method were also proven to be reasonable by comparison with high performance liquid chromatography (HPLC). Shimaa et al. screened two specific aptamers with dissociation constants of 2.8 nM and 2.7 nM for HbA1c and Hb, respectively, based on the systematic evolution of ligands by exponential enrichment (SELEX) process [52]. Then, the authors fixed the sulfhydryl-modified aptamers onto the surface of array electrodes modified by AuNPs to perform label-free detection of HbA1c and Hb. The sensor had a high sensitivity and detected HbA1c and Hb with detection limits of 0.2 ng/mL and 0.34 ng/mL, respectively. Such an array platform is superior to the existing immunoassay methods due to its simplicity, stability, low sample consumption, and low cost. However, the main disadvantage is that has a complicated operation. This method can detect HbA1c in human whole blood without any pretreatment and has broad applications in the diagnosis of diabetes.

Aptamer-type sensors can detect HbA1c specifically, but they usually require complex electrode modification. Shajaripour et al. proposed an electrochemical nano-genosensor, in which a reduced graphene oxide (RGO)-gold nanostructure was facilely electrodeposited on a graphite sheet (GS) electrode, and then, vulcanized DNA aptamers were fixed on the electrode surface [53]. The sensor had a high sensitivity of 269.2 µA/cm², a wide linear range of 1 nM–13.83 µM, and a low detection limit of 1 nM. This sensor has been successfully applied in blood samples and is expected to be a promising tool for diabetes screening and management. Furthermore, due to a large amount of carbohydrates and protein in whole blood samples, the effective antifouling ability of the electrode can improve the affinity and specificity of detection. Duanghathaipornsuk et al. [54] developed a gHb-targeted aptamer (GHA) through a modified SELEX process, and it was used to produce three distinct SAM-SPR-sensing surfaces with and without an antifouling layer. The results showed that the correlation between the HbA1c-targeted aptamer and HbA1c of the sensor surface with antifouling modification was higher than that of the sensor surface without modification, and the interference of nonspecific protein adsorption was reduced. This system illustrates the role of aptamers and antifouling surface modifications in developing effective, low-cost, and rapid HbA1c analyses in blood samples.

Antibody-based. Specificity and simplicity are the greatest advantages of this kind of immunosensor, which can satisfy the detection of HbA1c in a complex sample environment. Liu et al. fabricated a mixed layer on glassy carbon, which was attached by the redox probe 1,1′-di(aminomethyl)ferrocene (FDMA), followed by covalent attachment of the epitope glycated pentapeptide (GPP), an analogue to HbA1c, to promote competitive inhibition between antibodies and HbA1c [55]. A good linear relationship was observed between the relative faradaic current of FDMA and the concentration of HbA1c, ranging from 4.5% to 15.1% of the total hemoglobin in the serum, without the need for washing or rinsing steps. In addition, the preparation technology of antibodies is complex, time-consuming, and expensive. Karaşallı et al. [56] used a reduced graphene oxide (ERGO)-modified glassy carbon electrode (GCE) as a sensing interface and dropped an anti-HbA1c antibody solution onto the GC/ERGO electrode. A linear relationship was obtained between the DPV response and HbA1c concentrations from 1% to 25%. Alireza et al. [57] used a 3-mercaptopropionic acid (MPA) self-assembled monomolecular membrane to covalently attach anti-HbA1c antibodies. This binding process occurred on the gold electrode surface, which was previously coated with a polyethylene terephthalate (PET) substrate. For samples of HbA1c dissolved in 0.1 M PBS, this sensor had a dynamic range of 7.5–20 µg/mL. For undiluted human serum samples, a linear correlation was observed in the range of 0.1–0.25 mg/mL HbA1c. These results demonstrated that the sensor holds great potential in the treatment of diabetes in the future.

Catalytic-mimetic-based. In addition to boronic acid and its derivatives, ferrocene and its derivatives, nucleic acid aptamers, and antibodies, there are some other types of materials suitable as sensitive materials for fabricating electrochemical HbA1c sensors [58]. For example, flexible conductive artificial enzymes can be used for HbA1c detection. Conductive artificial enzyme nanoparticles were prepared by molecular imprinting technology [59]; in the presence of Rhodamine b and 3-aminophenyl boronic acid, Hb and HbA1c were embedded into the molecularly imprinted polymer, and then they were removed to form specific 3D binding sites in the polymer. The catalytic-mimetic HbA1c biosensor based on the lock-key model has good specificity and promotes the redox process. The linear ranges for HbA1c and Hb detection were 0.5–100 mM and 0.45–120 mM, respectively, and only 0.07 µL of the sample was required for one test.

Potentiometric HbA1c biosensors mostly include immune sensors based on integrated chips or extended gate electrode arrays as sensitive elements [60], and they can simultaneously detect Hb and HbA1c without markers. The integrated chip is built based on standard complementary metal oxide semiconductor (CMOS) technology. Before detection, anti-HbA1c antibodies are coupled with ion field effect transistors [61]. The detection mechanism of the potentiometric HbA1c sensor is that on the gate of the field effect transistor, the biofilm is deposited to form a double electric layer, and its potential changes along with the concentration of HbA1c [62,63].

Xue et al. built a potentiometric label-free immune microsensor using the CMOS process [63]. This sensor was composed of a CMOS with a micro-signal readout circuit and disposable test strip electrodes. Using a self-assembled monolayer film coated with AuNPs, the authors fixed antibodies onto the electrode surface and detected HbA1c and Hb within linear ranges of 4–24 mg/L and 60–180 mg/L, respectively. Furthermore, an improved AuNPs sensor with SAMs eliminated nonspecific sites and interference. Compared to an immunosensor fabricated by the mixed SAMs method and without gold nanofilm, this sensor had twofold higher sensitivity [64]. Another group built a disposable potentiometric immune sensor using screen-printed electrodes and PET [65]. To improve the sensitivity by exposing HbA1c to the antibodies, 0.2% ammonium dodecyl trimethyl bromide was added to denature HbA1c. This sensor was successfully applied in the direct determination of HbA1c, and the results showed a good correlation between the HbA1c standard and measured values.

Additionally, alizarin red s (ARS) can be used as an indicator of the redox reaction in the potential detection of HbA1c [66]. A negative redox potential shift was produced by binding PBA to ARS and HbA1c after a complex reaction with dialcohol-boric acid [67]. First, the potential of the ARS-PBA complex was negative. After competition between HbA1c and ARS to bind PBA, however, the potential shifted positively, and the shift value was related to the HbA1c concentration. The concentration of HbA1c measured according to the potential changes was in good agreement with the reference results.

Accurate and rapid detection has always been a research hotspot in the field of medical diagnosis. Impedance sensors are effective in detecting the reaction mechanism of the modified electrode interface and provide a fast detection method by studying the conductivity and chemical conversion process with electrochemistry [68]. In general, the detection mechanism of impedimetric HbA1c sensors is that the accumulation of HbA1c on the biosensor film changes the resistance characteristics of the electrode interface [68]. These sensors can detect affinity interactions (e.g., antibody-antigen interactions) without labeling in real time [69,70,71,72]. Park et al. reported a novel sensor with HbA1c immobilized on a gold electrode covered by an SAM of thiophene-3-boronic acid (T3BA), using K₃Fe(CN)₆ and K₄Fe(CN)₆ as a redox probe. The rate of charge transfer between the electrode and the redox probe is related to the concentration of HbA1c [73]. Since HbA1c is difficult to distribute evenly on the sensor surface, the stability of the detection results is not excellent. Although the redox agent can significantly improve electron transfer, it may also diminish the activity of the electrode/SAM interface over time.

Fortunately, Hu et al. found that the electrode could detect surface binding behavior within a specific frequency range, even without a special redox reagent [74]. Chuang et al. [75] achieved the detection of HbA1c without redox reagent in the frequency range of 20–1000 Hz. The sensor consisted of a pair of parallel electrodes integrated into a microfluidic device that was modified by an SAM of T3BA. This sensor can be easily integrated into a microfluidic device, consuming a low amount of the sample. Furthermore, Hsieh et al. [76] proposed a circular gold finger-like electrode on this basis, which still required no redox reagents. The electrode could measure HbA1c concentrations from 1 to 100 ng/µL at frequencies ranging from 0.5 to 20 kHz. This strategy makes it more suitable for POCT applications. Moreover, Boonyasit et al. proposed a novel 3D paper-based electrochemical impedance device combined with haptoglobin (Hp)-modified and APBA-modified eggshell membranes (ESMs) that was highly responsive within the clinically relevant total concentration range (0.5–20 g/dL) and that of HbA1c (2.3–14%) and reduced the data acquisition time 15-fold [77]. This micro-fast sensor not only shows great potential for POCT but is also a unique platform for off-site clinical diagnosis.