A Facile Colorimetric and Spectrophotometric Method for Sensitive Determination of Metformin in Human Serum Based on Citrate-Capped Gold Nanoparticles: Central Composite Design Optimization.

For the determination of Metformin in human serum, a facile colorimetric and spectrophotometric sensor was designed based on citrate-capped gold nanoparticles (citrate-GNPs). In this probe, the addition of Metformin to GNP solution generates a naked-eye color change resulting from the aggregation of GNPs. Study of this color conversion and quantity analysis of analyte is operated by spectrophotometric instruments. The three factors pH, time, and GNP ratio were selected to examine their effects on sensing results and their values optimization. The optimization of parameters was done by means of central composite design and one-at-a-time methods. The sensing results proved the highly selective and sensitive performance of the sensor for Metformin in a linear range of 6.25-133.3 ppm with a detection limit of 1.79 ppm. The relative standard deviation (RSD) of the reported method is 2.53%.

Introduction

Diabetes, a worldwide disease, is classified in two types: type 1 or insulin-dependent and type 2 or noninsulin-dependent. Based on reports published by International Diabetes Federation, the total number of those suffering from diabetes is growing every year. For instance, according to statistic predictions, type 2 diabetes, currently affecting about 8% of adult population, would spread in such a pace that by 2030, more than 40 million cases can be found throughout the world.[1−3] Metformin (N,N-dimethylbiguanide) is a popular glucose-lowering agent that helps type 2 diabetic patients in better insulin performance.[4−8]

Scientists have reported different diagnostic techniques for Metformin determination to study and monitor this drug in biological fluids. Most techniques are based on high-performance liquid chromatography-ultraviolet (HPLC-UV), potentiometry, liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS), capillary electrophoresis-ultraviolet (CE-UV), and thin layer chromatography (TLC). However, these methods have low sensitivity and a time-consuming sample pretreatment procedure is required.[1,9−11]

Gold nanoparticles (GNPs) have been used in the field of catalysis, metal ions, and biological molecule detection based on their optical properties, electrical properties, biocompatibility, and numerous extinction constants.[12−18] One well-known optical aspect of GNPs is localized surface plasmon resonance (LSPR) resulting from the interaction of conductive band electrons with electromagnetic waves of the incident light. This interaction results in the production of an LSPR with a frequency that depends on the factors such as dielectric media, geometry, and distance between nanoparticles.[19−25]

Aggregation in GNPs changes the interparticle distances and leads to a coupling and overlap between plasmon of nanoparticles, causing a red shift in LSPR bands and naked-eye color changes in solution.[20,21] This color changes can be used as a colorimetric sensor to determine different targets. There is no need for complex instruments, and the determination is performed via a simple optical tool.[20,26−30]

Therefore, in this study, we developed a simple and rapid colorimetric and spectrophotometric technique to determine Metformin in human serum without utilizing any pretreatment and dilution procedure in the serum sample with high selectivity and sensitivity. Upon the addition of Metformin to the citrate-GNP solution, an aggregation in nanoparticles occurred resulting in naked-eye color changes from red to gray and appearance of a new peak in visible spectrum. Effects of the three factors on Metformin sensing were investigated by central composite design (CCD) and one-at-a-time methods. The important parameters such as stability of sensing performance, selectivity, sensitivity, and interferences efficacy were measured. This optical sensor reported excellent findings for detecting Metformin in buffer and serum media.

Results and Discussion

Preparation of Citrate-Capped Gold Nanoparticles

Citrate-capped gold nanoparticles (citrate-GNPs) were synthesized in water via the citrate-mediated reduction of HAuCl4. First, an aqueous solution of 1 mM HAuCl4 was heated in a flask fitted with condenser. When the solution was boiled, 10 mL of 77.6 mM trisodium citrate solution was added to the above system instantly. Second, the mixed solution was heated for another 10 min under vigorous stirring. The solution’s color immediately changed from pale yellow to ruby red, which indicates GNP formation. Reduction of gold ions and capping of resulting nanoparticles, both occurred by citrate ions. Finally, the heating was stopped, and the solution was stirred until it reached room temperature. The final solution should be stored at 4 °C for future experiments without aggregation. The TEM image from the final solution proved the spherical shape and mean size of citrate-GNPs (∼12.5 nm) (Figure a,b).

Figure 2

(a) TEM image of gold nanoparticles (scale bar is 150 nm). (b) Diameter distribution of GNPs. (c) DLS of GNPs. (d) Size statistics report by volume from DLS. (e) Zeta potential of GNPs.

Measurements

The colorimetric determination of Metformin was operated as follows: 1.5 mL of aqueous citrate-GNP solution (GNP ratio is 0.3) was added to a glass cell, and 100 μL of the standard solution of Metformin (in BR buffer with pH = 6.3) was introduced to the above system. The mixture was shaken completely at room temperature. The color of solution changed from ruby red to gray, indicating the aggregation of GNPs in the presence of Metformin. For quantity analysis of Metformin concentration, the same process was gone through, and the visible spectra of GNPs were recorded from 400 to 800 nm, before and after the addition of Metformin.

Real Sample Analysis

To ensure the reliability of the present probe, the buffer (pH = 7) and serum were selected as real samples. The serum was collected from a medical laboratory and just filtered with a 0.2 μm filtering paper. A certain volume of Metformin standard solution (10 ppm to 1000 ppm) was spiked into buffer and serum and then mixed entirely. GNP solution (1.5 mL, GNP ratio is 0.3) was added to the cell, and 100 μL of the real sample was later added to the above system. The results were analyzed by means of a calibration curve previously plotted for Metformin in different concentrations. The recovery of the sensor was calculated as a result.

Data Analysis and Statistical Software

Both CCD and one-at-a-time methods were used to realize the effect of pH, GNP ratio, and time on sensing performance. CCD method at five-level was used to study the effect of two factors (pH and GNP ratio) on the response of the reported colorimetric sensor. These factors and their levels were selected based on our tests and previously published papers. The high and low levels of each factor are shown below (Table ). Design Expert software version 7 was used to design the 14 random tests in two blocks and analyze the data. The response of each test is the absorbance intensity in 645–655 nm (Table S1).One-at-a-time method was used to test the effect of time on the sensing performance.

Table 1

Experimental Factors and Levels in CCD

	Level
Independence variable	–α	–1 (low)	0	+1 (high)	+α
pH	3	4	6	8	9
GNP ratio	0.05	0.1	0.2	0.3	0.35

Optical Properties and Characterization of GNPs

The analyzed characteristics of synthesized citrate-GNPs are mentioned here. The synthesis of citrate-GNPs was confirmed by observing the LSPR absorbance peak at λmax = 525 nm (Figure ). Size and shape of GNPs are proved by the TEM image. The TEM image of the GNPs proved both the spherical shape and size distribution. The mean size is 12.5 nm (Figure a,b). Figure shows the DLS and zeta potential analyses of nanoparticles. The average diameter was 11.9 nm with a PDI value of 0.730. The zeta potential data revealed the information about the charge on the surface of GNPs, which was negative here (−34.4 mV), and proved the presence of citrate on GNPs.

Figure 1

LSPR spectra of citrate-GNPs in the range of 400–800 nm.

FT-IR analyses of neat trisodium citrate (blue line) and citrate-GNPs (red line) are presented in Figure . The broad band at 3000–3500 cm–1 in both spectra is related to the C–OH stretching vibration. The sharp peaks around 1399 and 1585 cm–1 are for the symmetric and asymmetric stretching of COO– in trisodium citrate. However, these peaks were observed in the FT-IR spectra of citrate-GNPs with a shift to highest values (∼1411 and 1585 cm–1) and indicate the formation of citrate group on the surface of a new compound (GNPs).

Figure 3

FT-IR spectra of trisodium citrate (blue line) and citrate-GNPs (red line) in the scanning range of 1300–3800 cm–1.

Optimization of Effective Parameters

The effects of each factor (pH and GNP ratio) on colorimetric performance of citrate-GNPs were studied by the CCD model. To test the validity of the CCD model, the effect of parameters, and the interaction between them, ANOVA analysis was applied.

The reliability of the predicted model was documented by R2 and adjusted R2 In the reported probe, R2 was 0.9598 and adjusted R2 was 0.9311, which indicated high correlation between the experimental result and the predicted model. Based on ANOVA data, F value of the model was 33.43, and P value less than 0.0001, which proved that the model was significant. The lack-of-fit (LOF) P value was (0.8842) not significant and confirmed the acceptability of the model (Table S2). The normal plot of residuals and interaction between parameters are illustrated in Figures S1 and S2, respectively. Most residuals are close to the line and indicate the excellent relationship between experimental results and the model. The R2 values approximate to 1. This plot shows the random distribution of the points.

The optimal parameters of sensing process are listed as follows: pH = 6.1 and GNP ratio = 0.3. These optimal points were used in all of the experiments.

The effect of time was studied by the one-at-a-time method. In a lower GNP ratio (0.1), the aggregation of GNPs occurred around 5 min after the addition of Metformin, as shown in Figure . But in a higher GNP ratio (0.35), the aggregation occurred in about 10 s. Optimal ratio of GNPs was 0.3 (based on result from the CCD models) so we chose 10 s as the optimum time for reaction.

Figure 4

Effect of time in the colorimetric performance of GNPs in two ratios (a) 0.1 and (b) 0.35.

Sensing Mechanism

The reported sensor was based on citrate-GNPs and acted as a medium for colorimetric detection of Metformin in buffer and human serum samples. Citrate-GNPs were synthesized via a simple and well-known one-step method with inexpensive primary substances.

Additions of Metformin to GNP solution lead to a rapid naked-eye color change from ruby red to gray. The change occurred as a result of the aggregation of citrate-GNPs in the presence of Metformin. The LSPR spectra of GNPs (λmax = 525 nm) decreased by the addition of Metformin, and a new peak around 645 nm appeared (Figure ). Citrate-GNPs were negatively charged due to the presence of citrate ions on their outer surface. These negative charges interacted strongly with positively charged amine groups of Metformin via electrostatic interaction to form a noncovalent conjugation between gold and Metformin. The citrate-GNPs came closer to interact with amine groups of Metformin, resulting in an aggregation in GNPs solution. The intensity of the peak around 645 nm was proportional to the concentration of Metformin.

Figure 5

Mechanism of sensing (a) visible spectra of Metformin (yellow) and GNPs before (red) and after (gray) addition of Metformin. (b) Colorimetric performance of GNPs.

.

Analytical Characteristics

The response of this sensor was tested by adding different concentrations of Metformin in optimal conditions reported by the software. Figure shows the relation between Abs and the Metformin concentrations under the optimal experimental condition. The inset of Figure shows that the Abs values have an appropriate linear correlation with the concentration of Metformin in the range of 6.25–133.3 ppm. The linear regression equation is y = 0.0062x + 0.2268, where x is the concentration of Metformin (ppm) with a corresponding regression coefficient of 0.9171 and signal-to-noise ratio of 3 (S/N = 3) criterion. The detection limit of this sensor is 1.79 ppm (Figure ), which possesses an excellent performance. Furthermore, the analytical efficiency of the reported citrate-GNP colorimetric probe for Metformin detection is comparable to those reported.

Figure 6

(a) LSPR spectra of GNPs after addition of Metformin in different concentrations. (b) Linear calibration plot of Abs versus Metformin concentration (ppm).

Selectivity and Repeatability

To test the selectivity of the reported colorimetric sensor, the effects of two groups were studied. The first group was the ions and the second one contained drugs and components that may exist in human serum. Selection of possibly interfering ion was based on components of real samples (serum), and the concentrations of these ions were selected upon their natural amount in healthy human serum. In these tests, a certain amount of each ion (Cl–, Na+, Mg2+, Fe3+, K+, Ca2+, H2PO4, and Cu2+), drugs (Ciprofloxacin (CIP), Ibuprofen, Atorvastatin, Glibenclamide, and Doxorubicin (DOX)), and components (methylene blue, fast green, glucose and fructose) were added to citrate-GNP solution, and their effects were examined according to the optimized procedures. Addition of some ions and components in both groups showed no color changes and could not affect the colorimetric performance of the sensor for Metformin. However, the others showed the same color changes as Metformin, so we recorded their visible spectra to understand their effects on the sensor. However, visible spectra of no one were the same as those of Metformin. As shown in Figure a,b, the chosen ions, drugs, and component possess no significant effect in the spectrophometric performance of the Metformin determination sensor. The results indicate that citrate-GNPs can be used as a selective colorimetric and spectrophotometric sensor for Metformin detection in real samples.

Figure 7

Effect of (a) interfering ions and (b) drugs and compound on the colorimetric sensing of Metformin.

To examine the repeatability of sensor results, two approaches were adopted, yet through the entire tests, the measurement steps, measuring apparatus, and working conditions were the same. First, we performed four experiments over a day but in different hours. Then, we repeated four experiments within four different days and calculated the relative standard deviation (RSD %) for each set. As shown in Figure , the RSD values are 1.04 and 1.33%, respectively. The RSD value is less than 5% and indicates an excellent repeatability of the reported system.

Figure 8

Repeatability of reported sensor (a) in 4 days (RSD = 1.04%) and (b) in a day (RSD = 1.33%)

Application in Real Samples

The presented sensing system was applied to confirm the high efficiency of the citrate-GNP sensing system in real samples; the sensor was tested in buffer and human serum samples via standard addition techniques. The recovery of Metformin for spiked samples was in the range of 96.36 to 99.61%, whereas the mean relative standard deviation was 2.87. The sensor results were listed in Table . The results showed that the designed sensor was selective and sensitive for Metformin detection in real samples.

Table 2

Analytical Results (N = 3) of Metformin in Buffer and Serum Samples

Sample	Spiked (ppm)	Found (ppm)	Recovery (%, n = 3)	RSD (%, n = 3)
Buffer (pH = 7)	0.000	NDa
	12.500	12.452	0.9961	3.13
	50.000	48.180	0.9636	3.60
Human serum	0.000	NDa
	25.000	24.220	0.9690	3.18
	75.000	72.740	0.9698	2.65

ND = Not detected.

Conclusions

The findings of this study prove citrate-capped GNPs can be used as a facile and low-cost sensor for colorimetric and spectrophotometric determination of Metformin in buffer and human serum samples with high selectivity and sensitivity. Citrate-GNPs were synthesized via a simple and well-known one-step method and used as a colorimetric base in this sensor. The presence of Metformin changed the color of GNP solution from red to gray, resulting from the aggregation of GNPs. The effects of selected parameters (pH, GNP ratio, and time) on the sensing performance were optimized by CCD and one-at-a-time methods. The experimental results matched the CCD model. The compatibility of the model was confirmed by LOF, F, and P values. Furthermore, the reported sensor can perfectly detect different amounts of Metformin in real samples. The proposed method did not need any sophisticated apparatus or sample preparation (Table ) and can be applied for routine analysis.

Table 3

Comparison of the Present Work with Other Reported Techniques for the Determination of Metformin

Methods	Linear range (μg/mL)	LOD (μg/mL)	Materials and equipment	Reference
Potentiometric	8.6–16.5 × 104	8.6	PVC membrane sensor	(5)
			Microprocessor
			Ionalyzer pH millivoltmeter
Reversed-phase liquid chromatography (LC)	4.0–12.0	0.019	C18 ODS column	(6)
			Acetonitrile–water–acetic acid as mobile phase
Hollow fiber liquid-phase microextraction	0.001–1.0	5.6 × 10–5	Dihexyl ether as organic solvent	(9)
			NaOH and derivatization solution (PFBC in acetonitrile) in the donor phase
			HCL as acceptor phase
Derivative spectrophotometric method	1.0–10.0	0.05		(10)
Capillary electrophoresis with electrospray mass spectrometry(CE-ESI-MS)		2.14 × 10–3	Formic acid was used as background electrolytes	(2)
			A composition of methanol/water/ formic acid used as Sheath liquid
High-performance liquid chromatography-tandem mass spectrometry(LC-MS-MS)		0.002	Phenomenex Synergi POLAR-RP 80A column	(1)
Colorimetric and spectrophotometric	6.25–133.33	1.79	GNPs	Present work
			UV–vis dual-beam spectrophotometer

Experimental Section

Materials

For the synthesis of GNPs and sensing procedures, chloroauric acids (HAuCl4), trisodium citrate, sodium chloride, magnesium chloride, iron(III) chloride, potassium chloride, calcium chloride, sodium dihydrogen phosphate, and copper(II) chloride were purchased from Merch Co. Methylene blue, fast green, D-glucose, and D-fructose were obtained from Sigma-Aldrich. Metformin was obtained from Mahban Co. (Iran, Tehran). Ibuprofen, CIP, and Atorvastatin was purchased from Pursina Co. (Iran, Tehran). Glibenclamide and DOX were purchased from Chemidarou Co. (Iran, Tehran) and Ebewe Pharma Co., respectively. Other chemicals obtained were of analytical grade and used without purification. Double-distilled water (DDW) was used for all experiments.

Apparatus

A PG Instrument T80+ UV–vis dual-beam spectrophotometer was utilized to record UV–vis absorption spectrum from 400 to 800 nm. The tests were totally performed in a matched 1.00 cm glass cell. Transmission electron microscopy (TEM) images were taken by a Philips-CM30 transmission electron microscopy operating under 300 kV voltages. The size distribution of GNPs was obtained by measuring the size of about 100 particles to obtain the average size and the data resulting from dynamic light scattering (DLS) from Malvern Co. The pH value was adjusted and measured by a Metrohm Co pH meter. Fourier transform infrared (FT-IR) spectrum was recorded via a Shimadzu-8400S spectrometer within the range of 400–4000 cm–1 at room temperature by using KBr disks.