Fourth‐generation glucose sensors composed of copper nanostructures for diabetes management: A critical review

Abstract More than five decades have been invested in understanding glucose biosensors. Yet, this immensely versatile field has continued to gain attention from the scientific world to better understand and diagnose diabetes. However, such extensive work done to improve glucose sensing devices has still not yielded desirable results. Drawbacks like the necessity of the invasive finger‐pricking step and the lack of optimization of diagnostic interventions still need to be considered to improve the testing process of diabetic patients. To upgrade the glucose‐sensing devices and reduce the number of intermediary steps during glucose measurement, fourth‐generation glucose sensors (FGGS) have been introduced. These sensors, made using robust electrocatalytic copper nanostructures, improve diagnostic efficiency and cost‐effectiveness. This review aims to present the essential scientific progress in copper nanostructure‐based FGGS in the past 10 years (2010 to present). After a short introduction, we presented the working principles of these sensors. We then highlighted the importance of copper nanostructures as advanced electrode materials to develop reliable real‐time FGGS. Finally, we cover the advantages, shortcomings, and prospects for developing highly sensitive, stable, and specific FGGS.


| INTRODUCTION
Glucose is the primary source of energy in living cells and plays a critical role in biology. Diabetes can result in elevated blood glucose levels that pose a severe hazard to human health. 1,2 Diabetes is an overgrowing global public disease and is characterized by insufficient insulin formation or distribution in the body, causing the death of 1.6 million people per year worldwide. [3][4][5][6] It is a chronic condition that requires daily monitoring of blood glucose levels, 7 and in severe cases, insufficient insulin levels can result in diabetic ketoacidosis, leading to seizures. 8 Diabetes complications can also include neuro and cardiovascular diseases in addition to kidney disorders 9 and new risks such as heart failure, kidney dysfunction, poor vision, nerve damage, and disability. [10][11][12] These complications often result due to poor blood glucose control. Regulated and routine blood glucose tests are necessary when coping with emergencies, including hypoglycemia (low blood sugar level). [13][14][15] Detecting glucose levels rapidly and reliably in clinical and biological samples remains a major challenge. 16,17 Limiting sugar consumption and continuously tracking blood glucose levels is critical to managing diabetes and can significantly reduce lifethreatening diabetes and provide sufferers with a healthy lifestyle. [18][19][20][21][22] Electrochemical glucose sensors [23][24][25] with high sensitivity, good selectivity, rapid test, low-cost, reliable, and accurate in situ detection, [26][27][28][29] have attracted great attention when compared to other sensing technologies like chemiluminescence, 30 surfaceenhanced Raman scattering, 31 mass spectrometry, 32 calorimetry, 33 fluorescence spectroscopy, 34,35 and optical sensors. 36 In addition, substantial efforts have been made to investigate glucose sensing in various potential fields such as the pharmaceutical industry, pathology, physiology, food processing, and bio-fermentation. 37 Glucose sensors account for approximately 85% of the biosensors industry because they represent the direct health consequences of diabetes, which affects over 400 million people worldwide. 38 Electrochemical sensors are used to monitor blood glucose levels rapidly. 41 These devices allow for real-time detection. Furthermore, continuous glucose monitors have been used to enable autonomous insulin delivery, where glucose measurements automatically adjust insulin delivery in closed-loop systems. In this manner, insulin can be administered to the patient in cases of hyperglycemia. 41 Enzymatic glucose sensors (EGS) are based on glucose oxidase or glucose dehydrogenase enzymes and exhibit a very high and reliable sensitivity. 42 However, some limitation of such sensors, including chemical and thermal conditions, instability, and relatively high complexity of the test samples. 43 Fluctuations in external factors like pH, humidity level, and temperature, and so on, hinder further exploration in the field of enzyme-based glucose biosensors. [44][45][46] Enzymes-based glucose sensors are divided into three significant generations. [47][48][49] The first generation requires free oxygen to immobilize the enzyme (GOx) on the electrode. Oxygen dependency of these sensors has limited applications in oxygen-deficient blood samples. 15,50 The second generation of enzyme-based glucose sensors included an artificial mediator, which directly reacts with the enzyme glucose oxidase leading to less sensitivity and accuracy. Artificial mediators involved one-electron reversible redox ferrocene derivatives and ferrocyanide. 51 The third generation was investigated to compensate for the shortcomings of the previous generations. However, minor changes in pH, temperature, and humidity were still susceptible to enzymatic denaturation. 3,12 The immobilization of enzymes on the conducting electrode's surface is complex, and its quantity cannot be precisely controlled. The high cost, complicated fabrication procedure, short shelf life, and poor reproducibility of enzyme-based glucose sensors have always been challenging for researchers. 52,53 A description of enzymatic glucose oxidation mechanisms, viewed as first-, second-, and third-generation sensors, is depicted in Figure 1. 3 The aforementioned disadvantages of EGS attracted researchers to develop fourth-generation metal-based enzyme-free glucose sensors (FGGS) 54-56 that oxidize glucose directly on the electrode surface. [57][58][59] FGGS that do not rely on enzymes have gained widespread attention 60 and are considered ideal for glucose analysis because of their low cost, efficient sensitivity, high selectivity, and good stability.

| WORKING PRINCIPLE OF FGGS
Among the electrochemical detection techniques, two basic methods, amperometry and potentiometry, have been widely used. 15,50 The potential difference between a reference electrode and a working electrode is determined in potentiometric sensors at zero applied currents. The potential of the working electrode varies with the concentration of glucose. It has been shown that these sensors can evaluate glucose concentrations of 10 M or higher (an average human's blood glucose level is in the range of 4-7 mM). 12 Nonenzymatic electrodes have recently been developed by combining various metals and metal nanoparticles (NPs), including metal/metal oxide and alloy composites, for high sensitivity and low detection limit of the FGGS. 64 Bimetallic NPs can also be used in FGGS due to their superior electronic properties and increased catalytic activity. Similarly, alloys and metal oxides can be employed because they improve glucose oxidation and reduce the poisoning in the sensing electrodes of the sensor. 43 The previous decade has seen extensive advancements in the working mechanisms and principles of FGGS. [61][62][63] Like metal oxidebased non-EGS (NEGS), the copper-based FGGS functions at varying pH. The functioning of the sensor depends on the stimulation of the metal oxide surface. This occurs in the vicinity of highly reactive hydroxide ions, which also serve the catalytic purpose during the oxidation of glucose molecules. Tian et al. developed the following mechanism for glucose sensing using copper oxide-based NEGS. 64 CuO þ OH À ! Cu OH ð Þ 2 þ e À , The mechanism of this reaction is based on the electrochemical function of copper oxide that changes its oxidation states during the reaction. 65,66 This is evident in the above-mentioned chemical reactions that occur in the FGGS. During the sensing process, as the voltage shifts, Cu 2+ cations present in CuO get oxidized to Cu 3+, and CuOOH is formed ( Figure 2). This then allows the oxidation of glucose F I G U R E 1 A description of the mechanisms of enzymatic glucose oxidation in first-, second-, third-, and fourth-generation glucose sensors 3

| Mechanism of FGGS
The CuO-based glucose sensors combined with gold NPs (AuNPs) and modified with CuO nanowires electrode (CuO NWs) gave a linear range of 0.5 μM to 5.9 mM and sensitivity of 4398.8 μA mM À1 cm À2 and a rapid response rate of 5 s. 68 The synergistic mechanism proposed for CuO in alkaline media requires oxides, hydroxides, and oxyhydroxides for the electrochemical oxidation of glucose. 69 The strong catalytic properties of Cu and its derivatives have been reported to accelerate glucose oxidation. 110 Cu in CuO is electrochemically oxidized to strong oxidizing species such as Cu(OH) À4 or CuOOH À . Thus, the +2 oxidation state changes to +3 111 : Cu(III) catalyzes glucose's oxidation into gluconolactone and hydrolyzed into gluconic acid, as shown in Figure 4. 112 The reduction of Cu(III) to Cu(II) can be demonstrated by oxidation and reduction peaks. Cu(III) is the most responsible medium for electron transfer compared to other valence Cu ions. The stability of the AuNPs modified CuO NWs electrode was also investigated for more than 10 days with an interval of 2 days, which showed comparatively better stability than a bare CuO NWs electrode. The high catalytic capability of CuO NWs/AuNPs compared to bare CuO NWs could be attributed to incorporating AuNPs on the surface of CuO NWs, which significantly enhances the surface volume ratio of the designed electrode. The reported glucose sensor's properties were highly effective and reliable in testing human blood. Because of its high sensitivity and low limit of detection (LOD), it is suitable for noninvasive glucose detection in saliva and urine.
Cu III ð ÞþGlucose þ e À ! Gluconolactone þ Cu II ð Þ, Gluconolactone ! Gluconic Acid: Operating principle for glucose sensing. When the device is on (top), V pH = À1 V, the Pd contact absorbs H + from the solution and increases its pH. At high pH, the Au/Co 3 O 4 contact is in its more reactive CoO 2 oxidized state. With V g = 0.5 V, the CoO 2 contact oxidizes glucose and the resulting I g is collected, which increases with increased glucose concentration. When the device is off (bottom), V pH = 0 V, the pH is at physiological values, typically pH 7, no sensing occurs from the Au/Co 3 O 4 and I g = 0 A. Reprinted with permission from Reference 134, Copyright @ 2019 (Nature)

F I G U R E 2 Reactions that occur in a copper-based fourthgeneration glucose sensors (FGGS)
The sensitivity, detection range, detection limit, and response time of FGGS composed of copper and copper oxide nanostructures are given in Table 1. and their ability to control the kinetics of the reaction. 76 However, Pt and its derivatives are costly, which limits its practical application. 77 The same has been observed with Au-and Pd-based NEGS. Therefore, widespread applications of noble metals have been hampered by disadvantages like low selectivity, high cost, toxicity, and metal scarcity, making their use impractical on a larger scale production. 73,74 In addition, metallic, 75 [103][104][105][106] ( Figure 5).

| Electrochemical detection of glucose using copper-based FGGS
Because of their outstanding chemical and thermal stability, various nanomaterials exhibit remarkable sensitivity and selectivity in glucose sensing. 82 Their electrochemical properties, high electrode catalytic activity, low cost, strength, natural abundance, nontoxicity, and environmentally friendly nature [107][108][109][110] have made Cu and its oxides a potential candidate for various applications such as photoelectric devices, gas sensing devices, lithium-ion batteries, and especially as electrochemical sensors due to their optical nature and electrical characteristics. [82][83][84] CuNPs are an effective electrode material for glucose detection and are extremely sensitive to glucose oxidation due to their excellent electrical conductivity. 85    graphene oxide that showed a rapid response rate as low as 6 s and a low detection limit of 0.19 μM in human blood and urine samples. 132 Hence, recently the scientific focus has shifted toward developing nanomaterials-based NEGS that provide better linear range and ease in operation. Nanomaterials also possess sizes equivalent to enzyme molecules that aid in their functionalization. Highly conductive carbon-based nanomaterials are the best choice for electro-oxidation of glucose; however, their stability is a significant concern. As a result, researchers have concluded that copper and its bimetallic nanomaterials have a promising potential for fostering and promoting Also, these sensors show a highly accurate correlation between glucose levels measured in interstitial fluids and blood when measured using the commercially available blood-glucose meter. This correlation has been observed in several recent studies, [134][135][136][137][138] and thus, such sensors can be potentially used in clinical applications for glucose measurements.
In addition, the biocompatibility and shelf life of copper-based FGGS depend on the morphology of the copper architectures and the attached functional groups. 138 This is especially true in the case of implantable sensors made using NPs. Immune reaction against NPs is inevitable; hence, such reactions can be controlled by monitoring the size and shape of the NPs that indirectly influence the attachment of neutrophils or macrophages to them. [139][140][141] For example, particles that are not spherical and are over 6 μm in diameter may exhibit lowered macrophage adhesions and, therefore, will have better functional viability within the system. 138 Hence, the physical features of the NPs, their chemical nature, like their surface chemistry, influence their biocompatibility and enhance the sensor's overall life. 139 Furthermore, there is no sophisticated control over the protective sheath, thickness, and pore size of the nanoporous layer that would allow FGGS to work on plasma, human serum, and blood when undiluted. Moreover, disturbances caused by various electro-active and electro-inactive chemical species must still be adjusted. 61 Therefore, although these sensors offer promising alternatives to traditional, invasive blood glucose monitoring; further works need to be done produce better electrode protective films in FGGS, before these sensors are made available commercially on a large scale. Nanozymes have also gained popularity in recent decades. These nanozymes are nanomaterials that possess properties akin to enzymes and have been extensively studied for sensing purposes. 150

CONFLICT OF INTEREST
The authors declare no potential conflict of interest. [Correction added on September 24, 2021 after first online publication: Contribution details of Hamid A. Bakshi has been added.]

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.