Nicotinamide-Functionalized Carbon Quantum Dot as New Sensing Platform for Portable Quantification of Vitamin B12 in Fluorescence, Uv-Vis and Smartphone Triple Mode

Development of an efficient, portable and simple nanosensor-based systems with reliable analytical performance for on-site monitoring of vitamin B12 (VB12) are still major problems and a challenging work for quality control of manufacturers. Herein, a new fluorescence, UV-Vis and smartphone triple mode nanosensors were designed for the simultaneous detection of VB12 with high sensitivity and accuracy. A novel nanosensor was synthesized through nicotinamide-functionalizing on carbon quantum dot (NA-CQDs) by an one-step microwave-assisted method with green approaches. The NA-CQDs as fluorescence nanosensor showed excellent fluorescence properties and wide linear ranges from 0.1–60 µM with the detection limits of 31.7 nM. Moreover, color changes of NA-CQDs induced by the VB12 could also be detected by UV-Vis spectrophotometer and inhouse-developed application installed on smartphone as a signal reader, simultanusly. The Red, Green and Blue (RGB) intensities of the colorimetric images of NA-CQDs/VB12 system which taken by smartphone's camera converted into quantitative values by the introduced application. A smartphone-integrated with NA-CQDs as colorimetric sensing platform displays good linear ranges (4.16 to 66.6 μM) for on-site determination of VB12 with detection limit of 1.40 μM. The method was successfully applied in the determination of VB12 in complex pharmaceutical supplement formulations without any sample pre-treatment and matrix interfering effects. The recovery results (96.52% to 105.10%) which are in agreement with the reference methods, demonstrating the capability of the smartphone-assisted colorimetric sensing platform in many on-site practical applications of quality controls.


Introduction
Vitamin B12 (VB12), also called as cyanocobalamin, plays a vital role in normal metabolism of enzymes, lipids and carbohydrates, nerve cell maintenance, DNA synthesis and red blood cell formation (1)(2)(3)(4). For adults, the recommended dietary allowance of VB12 are about 0.40-2.80 mg per day (5). The excessive VB12 can resulted in liver disease, asthma, neurotoxicity and kidney disorders. Moreover, deficiency of VB12 may lead to significant public health problems like neurological degeneration, memory loss, pernicious anemia and increases the risk of heart disease (6). Vitamin B12 is one of the essential water soluble ''B complex vitamins'' that cannot be produced by body cells, it can also be taken from foods like milk, meat, egg and pharmaceutical supplements in various dosage forms daily (7). As recommended by a healthcare practitioner, the deficiency of VB12 can often be reversed by consuming supplements or by fortified foods (8). At present there is great attention in accurately assessing the total dietary intake of vitamins B12 from all sources, especially vitamin supplements (tablets or injections). Therefore, there is an urgent need to develop an accurate, fast and portable method for on-site quantification of VB12 at the quality control of pharmaceutical companies.
At present, most quantification of vitamin B12 are conducted using high performance liquid chromatography (HPLC) (9), enzyme-linked immunosorbent assay (ELISA) (10), chemiluminescence (11), liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) (12), capillary electrophoresis and electrochemical methods (7,13). Generally, these laboratory-based methods are expensive both in reagent and services and requires professionally trained operators (14). Also, another limitation of laboratory-based methods is their time-consuming properties, which leads to factory production line wait for several hours for the test results in order to release the high-quality products. Therefore, the development of an efficient, affordable and simple nanosensor-based systems with sufficient analytical performance for accurate and fast monitoring of vitamin B12 content is necessary for the evaluation of supplement quality.
With the progress of quantum dots as advanced nonomaterials, they are considered as promising fluorescence sensing platform for quantification of VB12 (15,16). Compared with conventional semiconductor quantum dots, carbon-based quantum dots (CQDs) offer benefits such as highly tunable photoluminescence, chemical inertness, small size and photo-bleaching resistance and also low toxicity and biocompatibility (17,18). Up to now, among developed nanosensor for quantitative detection of VB12, fluorescence-based nanosensor using quantum dots have been reported more than the others (5,8,19). It should be noted that, most of the developed fluorescent assays of VB12 for many applications are still conducted with expensive instruments in laboratory and are not suitable for fast on-site detection of analytes.
in recent years, smartphone-based detection system integrated with bio/chemical sensors has shown promising progress in point of care (POC)/ Point of use (POU) testing approaches for various analytes. Because they are portable, ubiquitous and also produce rapid and simple quantitative results that can be interpreted by untrained personnel especially in resource-limited areas (14,(20)(21)(22)(23). To-date, one research teams have been reported smartphone platform for POC quantification of VB12 in blood using immonuassay-based lateral flow biosensors (24). These biosensors fabrication needs complicated antibody preparation technology and test strip assembly skill, which is not easy to achieve. Furthermore, in the present study, a novel nicotinamide-functionalized CQDs nanosensor were designed as fluorescence and colorimetric probes for simple and portable detection of VB12 simultaneously. NA-CQDs were synthesized by one-pot microwave-assisted hydrothermal method emploing nicotinamide as new sources of functionalized material on carbon quantum dots for the first time. Moreover, we have try to make new nanosensor more user friendly and portable by smartphone. But to our knowledge, no such work has been reported so far by the smartphone integrated with NA-CQDs nanosensors for the on-site monitoring of VB12 in pharmaceutical supplements. The NA-CQDs nanosensor exhibited dual response via both fluorescence and colorimetric change for on-site monitoring of VB12, simultaneously. In the RGB-based (red, green and blue color space) software, while the image is captured by the camera, simultaneous analysis can be done by the in-house developed android app and the quantitative results are displayed on the smartphone. Therefore, the portable smartphone-assisted colorimetric reader is promising to become a sensitive tool for quantitative colorimetric measurement and accurate onsite analysis of VB12 in many applications.

Materials and Instruments
All standards or chemical reagents used were of pharmaceutical and analytical grades. Mapping) and high-resolution TEM (FEI Tecnai F20 HR-TEM) microscope. The FT-IR spectrum of NA-CQDs were acquired using a Fourier transform infrared spectrometer (FT-IR Spectrum two, Perkin Elmer) from 400 to 4000 cm −1 .

Microwave-assissted synthesis of nicotinamide-functionalized CQDs
Nicotinamide-functionalized CQDs have been successfully synthesized via a facile hydrothermal treatment of citric acid as C source and nicotinamide as new source of N through microwaveassisted hydrothermal method. In a typical experiment citric acid monohydrate (1.68 g) and nicotinamide (2.92 g) were thoroughly dissolved in 10 mL of ultrapure water and stirred for 30 min. Then the solution was transferred into microwave radiation system with moderate temperature (80 W, 160 °C) for 20 min.

Development of RGB-based smartphone-app
In smartphone-assisted colorimetric sensing platform, the color intensities of the images taken by

FT-IR
The surface chemical groups of nicotinamide-functionalized CQDs were further characterized by FT-IR (Fig. 1A). For NA-CQDs, a broad absorption band was observed at 3000-3400 cm − The results obtained from FT-IR are in agreement with other characterization and clearly confirm that nicotinamide-functionalized CQDs are successfully synthesized.

SEM and EDX elemental mapping
The morphology and elemental composition of NA-CQDs were characterized by FE-SEM apparatus which was equipped with an energy-dispersive X-ray (EDX) spectroscope. Fig.1B and EDX analysis and mapping were also conducted to evaluate the detailed elemental composition and distribution. The data generated from EDX spectrum of NA-CQDs showed the following atomic percentages, respectively: 67.8 wt% (C), 11.1 wt% (O) and 21.1 wt% (N). In addition EDX mapping in the Fig. 1C, confirmed that successful and uniform excistence of N element in the structure of nicotinamide-functionalized CQDs.

HR-TEM
Morphological and size distribution of NA-CQDs were further obtained by high-resolution transmission electron microscopy (HR-TEM). HR-TEM image (Fig.1E) illustrate that, the average diameter of NA-CQDs was smaller than 10 nm.

Fluorescence sensing performance of NA-CQDs for VB12
The capability of NA-CQDs as a fluorescence nanoprobe for monitoring of VB12 was investigated. Figure CQDs nanosensor exhibit wide linear range, high sensitivity and low detection limit, which is lower than these of the recent reports (Table.1).

Smartphone-assisted colorimetric sensing platform for VB12
Taking advantage of good performance of smartphone-assissted colorimetric platform for sensing analytes, the NA-CQDs/ VB12 system was utilized for on-site monitoring of VB12. As shown in the Fig.3, the color change of the NA-CQDs probe from blue to red induced by VB12, which were quantified through RGB profiling in the range of 4.16-66.6 µM. As revealed in As illustrated in Fig. 4A, the absorption spectral overlap of VB12 with the fluorescence excitation and emission peak of nicotinamide-functionalized CQDs, meets the requirement for inner filter effect fluorescence quenching strategy for sensing of VB12. This reasonable assumption does fit well with the previous literature (18,26).
In the present study, to more clarify color change in the NA-CQDs/B12 system, the UV-visible absorption spectra of N-CQDs in the absence and presence of VB12 were investigated and the results illustrated in the Fig. 4B. As shown in Fig. In the absence of VB12, the NA-CQDs had a characteristic peak about 334 nm. In the presence of NA-CQDs, no shift in wavelength was observed in the absorption spectra of VB12(33 µg/ml) but the peak at about 360 nm increases after additions of VB12 (Fig. 4B). The addition of VB12 (0-133.2 µM) into the NA-CQDs solution gradually increases the intensity of absorption band at about 360 nm, which resulted in color change of NA-CQDs nanosensor (Fig. 4C).

Selectivity and interference studies for the detection of VB12
Various common substances in VB12 supplements formulation may interfere with the accuracy of B12 detection. Therefore, Vitamin B1, B5 and vitamin B6 were applied as competitive formulations, were also evaluated. Moreover, As shown in Fig. 5B, it was obvious that these coexisting excipients did not interfere with the detection of vitamin B12 (Fluorescence change <2 %). Therefore, the results demonstrate that the selectivity of nanosensor toward VB12 and the possibility of practical application of NA-CQDs nanosensor in the determination of VB12 in various pharmaceutical supplements formulation (Injection and tablet).

Real samples evaluation
The applicability of the NA-CQDs nanosensors for quantitative detection of B12 were evaluated in pharmaceutical VB12 supplements in various dosage forms (injection and Tablet) as real samples.  Fig.S2 Fig.S3 Fig.S4).

Conclusion
We have successfully designed a triple mode detection of B12 via novel nicotinamide-