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Ultrasensitive determination of ascorbic acid by using cobalt oxyhydroxide nanosheets to enhance the chemiluminescence of the luminol–H2O2 system

Ascorbic acid (AA) as an essential vitamin in the human body participates in various physiological reactions and plays a key role in many biochemical processes. Therefore, it is of vital importance to monitor and quantify AA in commercial tablets, beverages and food. In this work, a rapid and ultrasensitive chemiluminescence (CL) system for the detection of AA was developed, in which ultrathin cobalt oxyhydroxide (CoOOH) nanosheets were applied in the conventional luminol–H2O2 CL system. The results showed that ultrathin CoOOH nanosheets as a catalyzer remarkably improved the CL intensity of the CoOOH–luminol–H2O2 system, up to about 1400-fold. Under the optimized conditions, the CL inhibition efficiencies increase linearly with the concentrations of AA in the range of 1–500 pmol L−1, and the limit of detection was 39 fmol L−1. Moreover, the proposed CL system was successfully applied in the determination of AA in medicinal tablets with satisfactory results.


Introduction
Ascorbic acid (AA), also called vitamin C or ascorbate, is a common water soluble organic compound involved in many biological processes and metabolisms. 1 As an essential micronutrient, AA plays an essential role in human and animal metabolic process, 2,3 and it is oen used as an antioxidant added to food, beverages and medicinal preparations. 4 The shortage of AA results in scurvy, and the excessive intake of AA can lead to stomach convulsions, urinary stones and diarrhea. Therefore, the rapid, sensitive, and selective detection of AA levels is of signicance in the cases of medical assays and diagnosis.
Up to now, a wide variety of analytical techniques have been developed to detect and quantify ascorbic acid, such as uorimetry, 4-6 chromatography, 7,8 spectrometry, 9 colorimetry, 10,11 and electrochemistry. 12,13 Yan's group developed a CdTe QD uorescent probe for the selective detection of AA aer quenching by a certain amount of KMnO 4 with the limit of detection of 74 nmol L À1 . 14 Zhang et al. have constructed a highly sensitive, selective electrochemical sensor for the determination of AA via the combination of a functionalized multi-walled carbon nanotube layer with a thin molecularly imprinted polymer (MIP) lm. The integration of MIT in organic electrochemical transistor sensors (OECTs) is a reasonably effective route to improve the selectivity and applicability of OECTs in biosensors. 15 Although these methods are highly sensitive, most of them suffer disadvantages such as expensive cost, time-consuming, a complicated pretreatment process and the operation of sophisticated instruments, which limit their wide applications. Compared with other methods, chemiluminescence (CL) sensors do not need an excitation source and the corresponding spectrometric systems. Many drawbacks of uorescence sensors will be avoided including photobleaching and autouorescence. These advantages of CL sensors greatly improve the sensitivity. Conventional and primary CL systems including luminol, acridinium ester, AMPPT, potassium permanganate and Ce(IV) have got extensive development and applications. All these methods have been used for detecting AA such as luminol-K 3 Fe(CN) 6 -GNPs, 16 cerium(IV)-rhodamine B CL system, 17 luminol-H 2 O 2 -gold colloids system, 18 and Mg-Al-CO 3 LDHs-catalyzed ONOOH system. 19 The luminol-H 2 O 2 system, as a popular and classic CL reaction, has been extensively investigated. It can be catalyzed by metal ions, nanoparticles, graphene oxide and peroxidases. However, there are still some shortcomings of these catalysts, such as high toxicity and cost for metal ions and metal nanoparticles, complicated synthetic steps for graphene oxide, and low stability for peroxidases.
In recent years, cobalt oxyhydroxide (CoOOH) nanosheets are known by its rich electrical and catalytic properties, 20 which were explored in sensing of AA. For example, Li et al. developed a novel strategy on the basis of the specic reaction of CoOOH and AA for detection and imaging of AA in living cells and in vivo. 21 CoOOH nanoakes can quench the luminescence of persistent luminescence nanoparticles (PLNPs), the presence of AA can reduce CoOOH into Co 2+ and the luminescence was recovered. Recently, Cui et al. reported that under the weak acidic condition and in the absence of hydrogen peroxide, CoOOH nanosheets can catalyze the 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) to produce blue solution which could reduce the uorescence intensity of albumin-stabilized gold nanoclusters (BSA-AuNCs). The addition of ascorbic acid destroyed some of the CoOOH nanosheets and retarded the catalytic oxidation, in hence, less uorescence was reduced. 22 Yao's group proposed a novel label-free uorescent sensing system to detect alkaline phosphatase (ALP) which can catalyze L-ascorbic acid-2phosphate into AA based on chemical redox strategy to modulate the uorescence of nitrogen-doped graphene quantum dots (NGQDs). 23 It is worthy to note that all these are turn-on uorescence probes. 24 However, the limited quenching capability of CoOOH nanosheets and the deciency in uorescence resonance energy transfer between CoOOH nanosheets and uorescent materials led to the low sensitivity of these methods. In addition, in uorescence detection an external light source is required.
In this work, the enhancing CL of luminol-H 2 O 2 system was built for AA detection in the presence of CoOOH nanosheets. To the best of our knowledge, it is the rst time that CoOOH nanosheets were applied in CL system. The prepared ultrasensitive CoOOH nanosheets exhibited excellent catalytic property on CL system. On the basis of the mechanism demonstrated in Scheme 1 the enhancing CL method for AA detection was developed. The results showed that CoOOH nanosheets have a remarkably enhancing effect on the CL of luminol-H 2 O 2 system when its concentration is low. AA can reduce the CoOOH nanosheets to Co 2+ and inhibit the CL of luminol-H 2 O 2 enabling the ultrasensitive detection of AA with the limit of detection as 39 fmol L À1 . This work provides an ultrasensitive, simple and rapid route to construct CoOOH-luminol-H 2 O 2 CL sensor, which has been successfully applied to the detection of AA in medicinal tablets with satisfactory results. Due to the fascinating properties of the modied CL system including ultrasensitive, simple, low consumption and easy to operate, it was anticipated that in the near future the method would has promising applications in sensitive detection of target analytes.

Reagents and materials
Ascorbic acid (reduced form), luminol, hydrogen peroxide (H 2 O 2 ), cobalt chloride (CoCl 2 ), sodium hypochlorite (NaClO), amino acids, and Tris (hydroxymethyl) aminomethane (Tris-HCl) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Vitamin C tablets were purchased from BY-HEALTH Co., Ltd. (Zhuhai, China). A 10 À2 mol L À1 luminol solution was prepared by dissolving 177.1 mg of luminol in 100 mL of 0.1 mol L À1 NaOH solution and stocked at 4 C. A stock solution of 5 Â 10 À2 mol L À1 H 2 O 2 was prepared by diluting 1.5 mL of 30% (v/v) H 2 O 2 to 13.5 mL with ultrapure water. Standard solutions of ascorbic acid were prepared daily in ultrapure water. All other reagents were analytical grade without further treatment and purchased from Aladdin reagent Co., Ltd. (Shanghai, China). All solutions were prepared using ultrapure water generated from an ELGA Labwater system (US) with an electrical resistance $ 18.2 MU. Morphology images of CoOOH nanosheets were photographed by transmission electron microscope (JEOL 2100 plus, Japan) at an accelerating voltage of 200 kV. A drop of sample solution was placed on a copper grid that was let to dry before being transferred into the TEM sample chamber. Fourier Transform Infrared Spectrometer (FTIR) results were tested on the IRAffinity-1 (SHIMADZU, Japan). Chemiluminescence was measured on the Spark 10M Microplate Reader (Bio-Tek, Winooski, VT). The UV-vis absorption spectra were collected on a Cary 60 UV-vis spectrometer (Agilent Technologies, USA). The crystal structure was identied by XRD-6100 (SHIMADZU, Japan). Zeta-sizer Nano (Malvern) was used to measure hydrodynamic size and zeta potential CoOOH-nanosheets. All the pH was performed with a Leici PHS-3C meter (Leici, Shanghai, China).

Preparation of CoOOH nanosheets
CoOOH nanosheets were synthesized by following the previous articles with slight modications. 23 In brief, 3 mL of NaOH (1 mol L À1 ) was added to 20 mL of CoCl 2 (5 mmol L À1 ) solution, subsequently, sonicated for 1 min. Then, 500 mL of NaClO (0.9 mol L À1 ) was added to the mixture and sonicated for 10 min. Aer that the product was collected by centrifugation at 10 619 Â g for 15 min, washed with ultrapure water for three times and freeze drying for 12 h. Finally, the power of CoOOH nanosheets was redispersed in ultrapure water with the aid of ultrasonic.

CL measurements of AA
The chemiluminescence sensing of AA were all performed in Tris-HCl buffer (10 mmol L À1 , pH 9.5). Typically, 250 ng mL À1 Scheme 1 Schematic illustration for AA detection by using CoOOH enhanced luminol-H 2 O 2 CL system.
CoOOH nanosheets solution and ascorbic acid solutions (0-10 nmol L À1 ) were added into 130 mL of 10 mmol L À1 Tris-HCl buffer (pH 9.5) in each well of the microtiter plate. Then the pumps were washed for three times with 10 À4 mol L À1 luminol and 0.1 mol L À1 H 2 O 2 . Aerwards, luminol solution and H 2 O 2 solution with the ow injection rates of 200 mL s À1 were injected into the target microtiter plate well. The specic schematic diagram of ow injection was illustrated in Scheme 1. The CL signals were monitored by PMT adjacent to the CL ow-through column. The signals were imported to the computer for data acquisition. The relative CL intensity {(CL 0 À CL)/CL 0 } was used for calibration and quantication of AA, where CL 0 and CL represented the CL intensities with and without adding AA, respectively.

Selectivity
To investigate the selectivity of the proposed method, the response of the system to 10 pmol L À1 AA and 100 pmol L À1 potential interferences which were prepared in ultrawater were tested. The interferences we tested are including twelve different kinds of amino acids (Trp, Gly, Phe, Ile, Val, Asp, Arg, Tyr, Cys, His, Leu, Lys), seven kinds of electrolytes (ZnSO 4 , MgSO 4 , CaCl 2 , KCl, NaCl, AlCl 3 , CoCl 2 ), glucose, sucrose and dopamine (DA). Subsequently, the CL intensity of the interfering species was recorded by utilizing Spark 10M Reader.

Detection of AA in commercial tablets samples
In order to study the detection performance of the method in practical applications, commercial vitamin C tablets were investigated. The vitamin C tablet was crushed in a pestle and mortar. The crushed powder (1.0 g) was weighed accurately and dissolved in 50 mL ultrapure water with ultrasonication. Then the solution was centrifuged at 10 619 Â g for 15 min to remove the insoluble impurities. Aerwards, the resulting solution was diluted 10 7 fold with ultrapure water to make sure the AA concentration fall into the detection range. Then 20 mL AA (200 pmol L À1 , 1 nmol L À1 , and 2 nmol L À1 ) was mixed with 130 mL diluted tablet solutions in the appointed well of the microtiter plate, and 10 mL CoOOH nanosheets (2.5 mg mL À1 ) were added. The assay conditions used were the same as mentioned above for detecting AA in vitamin tablets.

Preparation and characterization of CoOOH nanosheets
Under the alkaline condition, CoCl 2 can react with NaClO to form ultrathin CoOOH nanosheets. To investigate the morphology of the CoOOH nanosheets, the TEM assay was performed. As shown in Fig. 1A, the CoOOH nanosheets had the typical hexagon morphology with the particles size around 80 to 100 nm. Simultaneously, the DLS investigated the size of the CoOOH nanosheets and indicated that the particle size of the prepared CoOOH nanosheets is uniform (Fig. 1B). The zeta potential of the CoOOH nanosheets was 15.02 mV, showing the positive charge of the nanosheets (Fig. S1 †), which was attributed to its innovative properties of high conductivity, morphology control nature of micrometer/nanometer scale. The power XRD patterns (Fig. 1C) reveal that the crystalline nature of the prepared CoOOH nanosheets is hexagonal system and highly crystalline in nature without any impurities. The characteristic peaks corresponding to the rhombohedral heterogenite structure which in agreement with the standard JCPDS 07-0169. 25 The UV-vis absorption spectra result of  luminol-H 2 O 2 system; luminol-CoOOH system; luminol-H 2 O 2 -Co 2+ (the concentration of Co 2+ is 2.5 Â 10 À7 g mL À1 ); luminol-H 2 O 2 -CoOOH system; luminol-H 2 O 2 -CoOOH-AA system (the concentration of AA is 100 nmol L À1 ). (B) The CoOOH enhanced luminol-H 2 O 2 CL system responsed to different concentrations of AA: the concentration of AA is 0, 10 À12 , 10 À11 , 10 À10 mol L À1 , respectively. Conditions: luminol: 10 À4 mol L À1 ; H 2 O 2 : 10 À2 mol L À1 ; NaOH: 0.01 mol L À1 ; CoOOH: 2.5 Â 10 À7 g mL À1 ; pH 9.5; Tris-HCl, 10 mmol L À1 ; flow velocity, pump A: 200 mL s À1 ; pump B: 200 mL s À1 . The error bar represents standard deviation of three replicate measurements.
CoOOH nanosheets is showed in Fig. 1D, which has an absorption around 420 nm. 22 The inserted image indicated that the CoOOH nanosheets solution has a good dispersibility and possess the Tyndall effect of the colloidal solution. The FTIR spectrum of CoOOH nanosheets (Fig. S2 †) has three characteristic peaks at 3472, 1622, and 615 in the range of 4000-500 cm À1 , corresponding to the bond stretching of the hydrogen-bonded hydroxyl group (-OH), the characteristic of the Co-O double bond, and the Co-O 2À complex in the oxide. 26 The AFM image shows that the particle size of CoOOH nanosheets is around 100 nm (Fig. S3 †), which is conrms the result of Fig. 1B. Therefore, all of these results suggest that our CoOOH nanosheets were successful prepared.
The feasibility and mechanism of CoOOH nanosheets enhanced luminol-H 2 O 2 CL system to detect AA In order to research the feasibility of the CoOOH enhanced luminol-H 2 O 2 CL system, we recorded the CL intensity of the individual luminol solution, luminol-H 2 O 2 system, luminol-CoOOH system, luminol-H 2 O 2 -Co 2+ system, luminol-H 2 O 2 -CoOOH, luminol-H 2 O 2 -CoOOH-AA system respectively ( Fig. 2A). The results claried that the ultrathin CoOOH nanosheets can signicantly enhance the luminescence intensity of the classic luminol-H 2 O 2 system, up to about 1400-fold. At the same time, compared to CoOOH, Co 2+ has a relative low enhancement effect on the luminol-H 2 O 2 system. From Fig. 2B we can observe that the CL intensity decreased with the increase of the concentration of AA, which can be reckoned as the luminol-H 2 O 2 -CoOOH system can detect AA successfully. The mechanism of luminol-H 2 O 2 system has been widely explored, it is generally believed that luminol loses a H + ion in alkaline solution and becomes a negatively charged group i.e. 3-aminophthalic acid ion with the oxidation of H 2 O 2 . The chemical energy generated in the oxidation process and was absorbed by 3-aminophthalic acid ions in an excited state. Then the valence electrons of the oxidation state are changed from the lowest vibrational level of the rst electron excited state of the single line to each of the different vibrational levels of the ground state, it will produce the maximum wavelength of 425 nm. In order to explore the possible mechanism of the CL system, we measured the luminescence spectra of luminol-H 2 O 2 and luminol-H 2 O 2 -CoOOH CL system by uorescence spectrometer. As shown in Fig. S4, † aer addition of CoOOH, the maximum wavelength of emission was still 425 nm, indicating that the luminophore of this system is still excited 3-aminophthalate ion. Therefore, the addition of CoOOH nanosheets did not lead to the generation of a new luminophor for the CL system. Thus, the enhanced CL signals may due to the possible peroxidise-like activity of CoOOH nanosheets. In Scheme 2 we proposed the possible mechanism of the luminol-H 2 O 2 -CoOOH CL system. Under alkaline condition, luminol can be transformed into luminol radial. It is possible that CoOOH nanosheets possess peroxidise-like activity could catalyze H 2 O 2 to generate active intermediates such as OHc and O 2 c À . Luminol radical reacts with active intermediates yield 3-aminophthalate anion and produce the maximum wavelength of 425 nm. The addition of AA can reduce CoOOH into Co 2+ , which inhibit the catalyze effect of transform H 2 O 2 into active intermediates. The results showed in Fig. 2 demonstrated that the conventional luminol-H 2 O 2 CL system based on CoOOH nanosheets can be successfully used to detect the concentration of AA.

Optimization of experimental conditions
To acquire credible and reliable results, we optimized different kinds of experimental conditions including the concentration of H 2 O 2 , the concentration of CoOOH, the optimum buffer, the buffer pH and the optimal ow velocity. The experimental result showed in Fig. S5 † indicates that when the concentration of H 2 O 2 was in the range of 0.00 to 0.1 mol L À1 , the CL signal increased and reaches the maximum value while the concentration is 0.01 mol L À1 , aer that the CL signal starts to decrease. Therefore, 0.01 mol L À1 was chose as the best concentration of H 2 O 2 . Fig. S6 † showed that the CL intensity is the largest when the concentration of CoOOH is 250 ng mL À1 , so we chose 250 ng mL À1 as the optimal concentration of Scheme 2 Possible mechanism of luminol-H 2 O 2 -CoOOH CL system. CoOOH. In order to investigate the effect of buffer on the system CL intensity we chose ve different kinds of buffer solutions, we can observed from Fig. 3A that while the buffer is Tris-HCl the CL signal is the highest, so we chose Tris-HCl as the appropriate buffer for the CL system. We also investigated the pH of buffer, Fig. 3B illustrated that when the Tris-HCl buffer is 9.5 the CL intensity is the highest. Thus, 9.5 was chosen as the optimal pH in the following experiments. As for the ow velocity, we can observe from Fig. S7 † that there is no signicant variation of CL intensity between different ow rates. As a result, we nally chose the default value (pump A: 200 mL s À1 ; pump B: 200 mL s À1 ) as the optimal ow velocity for the followup experiments.

Detection of ascorbic acid based on CoOOH nanosheets enhanced luminol-H 2 O 2 CL system
In order to ascertain the application of the CL system to quantify ascorbic acid, under the optimal experimental conditions we measured the CL response of CoOOH enhanced luminol-H 2 O 2 system aer reaction with ascorbic acid. Fig. 4 shows the relative CL intensity against AA concentration, we observed that when the concentration of AA attains to 500 pmol L À1 the CL intensity intended to be steady, which means the sensing recovery reached the maximum. Moreover, a good linear relationship between (CL 0 À CL)/CL 0 and AA concentration which ranging from 1 pmol L À1 to 500 pmol L À1 with a correlation coefficient square of 0.9939 was obtained (inset in Fig. 4), where CL 0 and CL represented the absence and presence of AA, respectively. In addition, we also describe a comparison about the performance of this approach with previously recommended methods for AA determination in ESI Table S1. † The result indicates that the sensitivity is higher than many previous methods. Furthermore, the CoOOH nanosheets enhanced luminol-H 2 O 2 CL system can be applied to the determination of AA in various real samples.

Selectivity of CoOOH nanosheets enhanced luminol-H 2 O 2 CL system for AA detection
Selectivity is an important factor to evaluate the CL system for detecting AA, because of the complexity of the tested environment, multitudinous coexistent such as common amino acids, electrolytes and other biological molecules would have a negative effect on the AA detection. Several interfering substances including amino acids (Trp, Gly, Phe, Ile, Val, Asp, Arg, Tyr, Cys, His, Leu, Lys), electrolytes (ZnSO 4 , MgSO 4 , CaCl 2 , KCl, NaCl, AlCl 3 , CoCl 2 ) and some small biomolecules (glucose, sucrose and dopamine) were assessed under the optimal experimental conditions. As shown in Fig. 5, whether amino acids, electrolytes or small biological molecules do not generate an obvious and apparent interference on the proposed luminescence system even though the concentration of these interferents is 10 fold higher than that of AA. Therefore, this CL system based on CoOOH nanosheets could be applied for detecting AA in medicinal tablets or food samples directly.

Detection of ascorbic acid in commercial tablets
In order to explore the feasibility of this proposed procedure in complicated real sample environment, the assay was then Fig. 4 Plot of relative CL intensity against AA concentration (inset: linear relationship between (CL 0 À CL)/CL 0 and log concentration of AA). Conditions: luminol, 1 Â 10 À4 mol L À1 ; H 2 O 2 , 0.01 mol L À1 ; Tris-HCl 10 mmol L À1 ; pH 9.5; CoOOH, 2.5 Â 10 À7 g mL À1 ; flow velocity, pump A: 200 mL s À1 ; pump B: 200 mL s À1 . CL 0 represents the CL intensity without AA, and CL represents the CL intensity with different concentrations of AA. The error bar represents standard deviation of three replicate measurements. Fig. 5 Relative CL intensity of CoOOH enhanced luminol-H 2 O 2 CL system in the presence of 100 pmol L À1 potential interferents and 10 pmol L À1 AA. From left to right: blank, tryptophan, glycine, phenylalanine, isoleucine, valine, aspartic acid, arginine, tyrosine, tyrosine, histidine, leucine, lysine, glucose, sucrose, dopamine, ZnSO 4 , MgSO 4 , CaCl 2 , KCl, NaCl, AlCl 3 , CoCl 2 , AA. Conditions: luminol, 1 Â 10 À4 mol L À1 ; H 2 O 2 , 0.01 mol L À1 ; Tris-HCl 10 mmol L À1 ; pH 9.5; CoOOH, 2.5 Â 10 À7 g mL À1 ; flow velocity, pump A: 200 mL s À1 ; pump B: 200 mL s À1 . The error bar represents standard deviation of three replicate measurements. applied to detect AA spiked in diluted medical tablets. The tablets were dissolved, diluted and then spiked into three different kinds of concentration of AA. The content of AA in tablets was determined by this proposed method. As shown in Table 1, the recoveries of the spiked amounts of AA in diluted medical tablets solutions were obtained in the range of 95.0-98.6% with relative standard deviations (RSDs) below 1.56% for the real sample. These results proved that this CL sensing system based on CoOOH nanosheets-luminol-H 2 O 2 was likely to be capable of real sample analysis.

Conclusions
In this work, the ultrathin CoOOH nanosheets were rstly found which hold the ability of enchancing the chemiluminescence performance of the conventional luminol-H 2 O 2 system. The enhanced CL ability was ascribed to the possible peroxidise-like activity of CoOOH nanosheets. Based on this principle, this method shows strong detection ability for AA with ultrasensitivity and good selectivity. Meanwhile, this assay could not only function in aqueous solution for AA detection but also exhibit reliable responses toward AA in real samples. Taking full advantages of CoOOH nanosheets-luminol-H 2 O 2 CL system, this assay shows remarkable properties including ultrasensitive, fast, simple, and cost-effective as well as environmental-friendly, which suggest that this strategy has a great potential to be utilized as an efficient tool for determination of AA level in biological environments.

Conflicts of interest
There are no conicts to declare.