intrinsic fluorescence spectra characteristics of riboflavin and … · yang hui et al: intrinsic...

YANG HUI et al: INTRINSIC FLUORESCENCE SPECTRA CHARACTERISTICS OF RIBOFLAVIN AND NADH

DOI 10.5013/IJSSST.a.17.44.05 5.1 ISSN: 1473-804x online, 1473-8031 print

Intrinsic Fluorescence Spectra Characteristics of Riboflavin and NADH

Yang HUI*1, Xiao XUE2, Xuesong ZHAO2, Wu YAN1, Peibin CHEN1

1.New Star Application Technology Institute, Hefei, Anhui 230031, China

2.Key Lab. of Environmental Optics & Technology, AIOFM, CAS, Hefei, Anhui 230031, China

Abstract — The intrinsic fluorescence characteristics of NADH and Riboflavin/ Vitamin B2 with fluorescence Spectrophotometer were presented in this paper. Three strong fluorescence areas of Vitamin B2 locate at λex/λem=270/525nm, 370/525nm and 450/525nm and two fluorescence areas of NADH locate atλex/λem=260/460nm and 350/460nm are found. The influence of pH of solution value are also discussed, and with the PARAFAC algorithm, 9 Vitamin B2 and NADH mixed solutions are successfully decomposed, and the emission profiles, excitation profiles and the concentration of the two components are retrieved by about 10 iteration times. Vitamin B2 is a much stronger fluorophore with higher fluorescence efficiency than NADH, so low concentration of NADH in the mixed solutions affected the emission and excitation profile of NADH retrieved by the PARAFAC algorithm.

Keywords - NADH, Riboflavin/ Vitamin B2, Fluorescence Spectra, PARAFAC

I. INTRODUCTION

The fluorescence of a folded protein or bio-aerosol is a mixture of the fluorescence from individual aromatic component and coenzyme. Riboflavin, known as vitamin B2, is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. As such, vitamin B2 is required for a wide variety of cellular processes. Like the other B vitamins, it plays a key role in energy metabolism, and is required for the metabolism of fats, ketone bodies, carbohydrates, and proteins [1]. NADH is one of the most important coenzymes of the intermediary metabolism. During the processes ranging from digestion to the eventual synthesis of ATP, it is in charge of capturing free electrons and transporting them to the site of reaction. The critical role of NADH as the primary source of reducing equivalents is most prominent in the Krebs cycle and in oxidative phosphorylation. The signal emitted is proportional to intracellular NADH concentration.

Some previous studies reported that Parallel factor analysis, PARAFAC was applied to resolve the mixed and overlapped spectra of vitamin B1, B2 and B6 [2], accordingly, in this paper, discussed not only the fluorescence spectra of vitamin B2 and NADH individually, but also the overlap of fluorescence spectra of vitamin B2 and NADH at the Ex/Em area of wavelength within 240~370/470~500nm, by means of PARAFAC method.

PARAFAC, a three way-decomposition method, has been found to be very useful in identifying the independent spectra of different types of fluorophores [3]. Compared to its predecessor, Principal Component Analysis (PCA) technique, PARAFAC provides both a quantitative and qualitative model of the data and separates the complex signal measured into its individual underlying fluorescent phenomena with specific excitation and emission spectra. It can track even small variations in EEM datasets by separating several independent groups of

fluorophores from the overlapped components with a high resolution, so it is commonly used technique to monitor the mixed fluorescence EEMs. On the other hand, the weakness of PARAFAC model may include the assumption of the independence among the estimated components in the model, and potential inclusion of one or more poorly estimated components, which may significantly affect the spectra and scores of all other components [4].

II. EXPERIMENTAL SECTION

A. Instruments and Reagents

The Molecular ΣH2O ultra pure water machine (Shanghai Molecular Co. Ltd) was used to generate the ultra purified water, UPW whose pH value is 5.4. The riboflavin (vitamin B2) and NADH mother liquid were compounded with Riboflavin from Amresco co.Ltd whose purity greater than or equal to 98%, and the nicotinamide adenine dinucleotide, NADH from Xi’an BaiChuan Biotech co.Ltd with purity greater than or equal to 99.5%. The reagents were weighed with Mettler Toledo precise electronic balance, and dissolved with Briton Robson Buffer with different pH values (1.95, 5.4, 5.66, 8.0 and 11.92).

The vitamin B2 and NADH mother liquid concentration are 10mg/L and 25mg/L respectively. The vitamin B2 and NADH mother reagent solutions were transferred through DragonLab whole disinfection manual single channel adjustable liquid shifter and dilute to working solutions of different concentrations. All solvents were of analytical grade, all solutions and put in amber glass bottles and stored in a refrigerator (4 ) because of the light sensitivity of riboflavin.

3D fluorescence intensity measurements were carried out on an F-7000 FL spectrophotometer (Hitachi High-Technologies Corporation, Japan).



B. Instrument Settings and Experiment Procedure

500ul Briton Robson Buffers with different pH values and mother liquids of different volumes were injected into the 10ml test tubes, and diluted with purified water to form the working liquids and background liquids.

For the fluorescence EEM measurements of vitamin B2, the spectrophotometer excitation wavelength ranged from 200.0nm, to 550.0nm, emission wavelength ranged from 450.0nm to 650.0nm, scan speed was set at 12000nm/min with excitation and emission sampling interval of 10.0 nm, excitation and emission slit of 5.0nm, the PMT voltage was set at 700 V. Accordingly, for fluorescence EEM of NADH, the excitation wavelength ranged from 220.0nm, to 460.0nm, emission wavelength ranged from 350.0nm to 600.0nm. All experiments were performed at room temperature at 25 .

The 1st level and 2nd level Rayleigh scattering, Raman scattering and background components within the fluorescence signals were subtracted for the following analysis.

C. Principle of Multi-components Discrimination Using

PARAFAC Method

Based on the tri-linear decomposition theory, the PARAFAC method is a kind of mathematical model implemented through alternating least squares algorithm, which is widely applied to analyze three-dimensional or multi-dimensional data, to decompose N-dimensional data to the N load matrixes.

The measured fluorescence spectrum EEM data is a KJI matrix, in which, I indicates the number of the

samples, while J and K are the number of excitation wavelengths and emission wavelengths of samples respectively. Using Parallel Factor decomposition model, the fluorescence spectrum data matrix can be decomposed to score matrix A, load matrix B and C. The decomposition model can be represented as

ijk

F

fkfjfifijk cbx

1

（1）

where, i=1, 2, , I, j=1, 2, , J, k=1, 2, , K.

ijkx is the fluorescence intensity of sample i at excitation

wavelength j and emission wavelength k, F is the column

number of load matrix, or the number of factors, ijk is

the residual element, if , jfb , kfc are the elements in

load matrix A, B and C respectively. The algorithm will be aborted until convergence of the PARAFAC model, that

is, the minimum loss function

I

i

J

j

K

k

jkiSSR ef1 1 1

2 <10-6.

In this study, PARAFAC modeling was performed using the MATLAB 7.0 code. The appropriate number of components was determined primarily based on the three diagnostic tools including residual analysis, core consistency and visual inspection of spectral shapes of each component, which are widely used by other similar studies. The components extracted by PARAFAC represent groups of the organic components that exhibit

similar fluorescence properties. The component scores indicate the relative concentration of the groups, not the actual concentration of a particular material/fluorophore. However, it is typically assumed that the scores are proportional to the concentrations of the different components [5, 6].

III. FLUORESCENCE EEM CHARACTERISTICS OF

RIBOFLAVIN AND NADH

A. Intrinsic Fluorescence EEM Characteristics of

Riboflavin and NADH

For vitamin B2, there are three strong fluorescence areas, whose center locate at λex/λem=270/525nm, 370/525nm and 450/525nm respectively, and the emission wavelength ranges from about 500nm to 600nm, as shown in Figure 1. The fluorescence intensity excited by 270nm excitation wavelength is much stronger than that by 370nm and 450nm, the ratio of fluorescence intensity is 1:0.41:0.25 approximately.

Fig.1 Fluorescence Intensity Distribution of Riboflavin@60ug/L Accordingly, the NADH whose fluorescence

efficiency is about 0.024, has only two strong fluorescence areas, whose center locate at λex/λem=260/460nm and 350/460nm respectively, and the emission wavelength

b)

a)



ranges from about 420nm to 520nm, as shown in figure 2. The fluorescence intensity excited by 350nm excitation wavelength is much stronger than that by 260nm. The ratio of fluorescence intensity is 1.29:1 approximately.

Fig.2 Fluorescence intensity distribution of [email protected]/L

From figure 1 and figure 2, it can be seen that the fluorescence efficiency of vitamin B2 is much stronger than that of NADH for the same resolvent.

B. Fluorescence Emission Distribution of Riboflavin at

Three Excitation Wavelengths

1) Fluorescence emission distribution of vitamin B2 at excitation wavelength@270nm

450 500 550 600 650

0

2000

4000

6000

8000

10000

Excitation Wavelength@270nm

Flu

ores

cen

ce In

tens

ity/a

.u.

Emission Wavelength/nm

60ug/ml 100ug/ml 120ug/ml 200ug/ml 400ug/ml 500ug/ml 600ug/ml 800ug/ml 1000ug/ml

(a)

0 100 200 300 400 5001000

2000

3000

4000

5000

6000

7000

8000

9000

Flu

ore

sce

nce

Inte

nsi

ty/a

.u.

Concentration (ppb)

270nm Linear Fit at 270nm

Equation y = a + b*x

Adj. R-Square 0.97388

Value Standard Error

260nm Intercept 758.73391 365.56463

260nm Slope 17.73232 1.29517

(b)

Fig.3 Fluorescence intensity distribution of vitamin B2@270nm

The fluorescence intensity has a linear relationship with concentration when the concentration is less than 600ug/ml, and is saturated when concentration is over 600ug/ml

2) Fluorescence emission distribution of vitamin B2 at excitation wavelength@370nm

450 500 550 600 650-500

0500

1000150020002500300035004000450050005500600065007000750080008500 60ug/ml

100ug/ml 120ug/ml 200ug/ml 400ug/ml 500ug/ml 600ug/ml 800ug/ml 1000ug/ml


Flu

ore

scen

ce In

ten

sity

/a.u

.


(a)

a)

b)



0 200 400 600 800 1000

0

1000

2000

3000

4000

5000

6000

7000

8000

Flu

ore

sce

nce

Inte

nsi

ty/a

.u.

Concentration (ppb)

370nm Linear Fit @370nm




350nm Intercept 281.22264 180.72826

350nm Slope 7.31709 0.34443

(b)

Fig.4 Fluorescence intensity distribution of Vitamin B2@370nm

The fluorescence intensity has a linear relationship with concentration when the concentration is less than 1000ug/ml.

3) Fluorescence emission distribution of Vitamin B2 at excitation wavelength@450nm

450 500 550 600 650-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

60ug/ml 100ug/ml 120ug/ml 200ug/ml 400ug/ml 500ug/ml 600ug/ml 800ug/ml 1000ug/ml


Flu

ores

cenc

e In

tens

ity/a

.u.


(a)

0 200 400 600 800 10000

2000

4000

6000

8000

10000

Flu

ore

sce

nce

Inte

nsi

ty/a

.u.

Concentration (ppb)

450nm Linear Fit of 450nm




450nm Intercept 606.44099 184.87947

450nm Slope 9.57866 0.35234

(b)

Fig.5 Fluorescence intensity distribution of vitamin B2@450nm.

The fluorescence intensity has a linear relationship

with concentration when the concentration is less than

800ug/ml. From figure 3 to 5, it can be seen that the fluorescence

intensity is linear proportional to the concentration of fluorophore, and the strongest is excited by 270nm excitation than the other two excitation wavelength. Meanwhile, with the rise of the concentration, the collision of fluorophore molecular is increased, the energy loss through the non-radiative transition increases, the energy by radiative transition decreases, more energy quenched by the solution, and the energy emitted by the photon decreases, so the emission wavelength raises, accordingly, fluorescence intensity peak shifts to longer wavelength (red shift). The red shift of emission wavelength was also observed similarly by Ge[7].

C. Affection of pH to the Fluorescence Emission

Intensity Distribution

450 500 550 600 650

0

500

1000

1500

2000

2500

3000

3500

4000 PH1.95 PH5.66 PH5.4 PH8.0 PH11.92

Riboflavin 200ppb@260nmF

luo

resc

ence

Inte

nsity

/a.u

.


(a)

450 500 550 600 650

0

200

400

600

800

1000

1200

1400

1600

1800

2000 PH1.95 PH5.66 PH5.4 PH8.0 PH11.92

Riboflavin 200ppb@350nm

Flu

ore

scen

ce In

ten

sity

/a.u

.


(b)



450 500 550 600 650

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800 PH1.95 PH5.66 PH5.4 PH8.0 PH11.92

Riboflavin 200ppb@450nm

Flu

ore

scen

ce In

tens

ity/a

.u.

EmissionWaveleng (nm)

(c)

Fig.6 Fluorescence intensity distribution of vitamin B2@260nm,350nm

and 450nm affected by pH value of the resolvent.

When resolved in strong alkaline and strong acid solution, the structure of vitamin B2 molecular is distorted, so the fluorescence intensity declines sharply compared to the weak alkaline and weak acid solution, and if resolved in weak alkaline and weak acid solution, the fluorescence intensity are much stronger(seen in figure 6).

IV. ANALYSIS OF FLUORESCENCE SPECTRA OF

VITAMIN B2 AND NADH MIXED SOLUTION

A. Instrument Settings

For the fluorescence EEM Matrixes decompose of the mixed solution of vitamin B2 and NADH, the spectrophotometer excitation wavelength ranged from 200.0nm, to 550.0nm, emission wavelength ranged from 450.0nm to 650.0nm, scan speed was set at 12000nm/min with excitation and emission sampling interval of 5.0 nm, excitation and emission slit of 5.0nm, the PMT voltage was set at 700 V.

B. 3D Fluorescence Spectrogram of Riboflavin and

NADH Mixed Solutions

Fluorescence EEM intensity of vitamin B2 and NADH mixed solutions of different concentrations (listed in table1) are shown in Figure 7 (a) ~ (i).

TABLE 1. ANALYTICAL CONCENTRATIONS OF VITAMIN B2 AND NADH

Sample

Component

NADH Riboflavin

Original

(ug/L)

Retrieved

score

Original

(ug/L)

Retrieved

score

（1） 50 450 60 670

（2） 50 430 100 910

（3） 50 530 200 1910

（4） 50 470 500 5210

（5） 100 1090 60 530

（6） 100 890 200 1910

（7） 500 5050 60 550

（8） 500 4800 400 4310

（9） 500 5150 500 4910

a)

b)



c)

d)

e)

f)

g)

h)



Fig.7 Fluorescence EEM intensity distribution of vitamin B2 and

NADH mixed solutions of different concentrations.

C. Results Retrieved by PARAFAC Algorithm

Fig.8 Excitation and emission spectral profile retrieved by PARAFAC

algorithm, (a)Emission spectra, (b) Excitation spectra and (c)

concentrations of original solutions and retrieved, and (d) SSR and

iteration number

The emission spectra retrieved by PARAFAC are shown in Figure 8(a), the blue line with circle symbol (sample1) indicates the retrieved emission profile of vitamin B2 and the green line with block symbol (sample2) indicates the retrieved emission profile of NADH. From figure 8(a) and figure 1 to figure 5, one can see that the retrieved vitamin B2 emission profile and central wavelength are coincident with its real emission profile. But, for the retrieved green emission profile of NADH, the red shift of central wavelength was observed, this is probable induced by the low concentration and low fluorescence efficiency of NADH in the mixed solutions.

The excitation spectra retrieved by PARAFAC are show in Figure 8(b), the blue line with circle symbol (sample1) indicates the excitation profile of vitamin B2 and the green line with block symbol (sample2) indicates the excitation profile of NADH. From figure 8(a) and figure 1, it can be seen that the retrieved excitation profile and central wavelength are coincident with its real excitation profile. But for the retrieved green excitation profile of NADH, except the wavelength at @240nm and @350nm, the two @270nm and @450nm excitation wavelengths are observed, the extra two excitation wavelengths are induced by the relative high concentration and fluorescence efficiency of vitamin B2. The emission

i)

a)

c)



and excitation spectra of NADH are both influenced by that of vitamin B2.

The good linear correlations of original and retrieved concentration of NADH and vitamin B2 can be observed in Figure 8(c). From Figure 8(d) one can see that SSR of PARAFAC logarithms decreases quickly and sharply at the beginning of the iteration times, the SSR is stable when iteration times≥10.

V. CONCLUSIONS

The fluorescence of a protein or bio-aerosol or bio-agent is a mixture of the fluorescence from individual aromatic residues and coenzyme. Using fluorescence Spectrophotometer, the intrinsic fluorescent characteristics of NADH and vitamin B2 are measured with solutions of different pH and discussed. Vitamin B2 and NADH mixed solutions are successfully decomposed and resolved by PARAFAC algorithm. However, affected by the low fluorescence efficiency of NADH and concentration differences between the two fluorophores, the slight deviation of emission and excitation profile of NADH retrieved by the PARAFAC algorithm was observed.

ACKNOWLEDGMENTS

The work was supported by the National Natural

Science Foundation of China named “Study on

Technology of Ultraviolet Laser-induced Fluorescence

LIDAR for Bioagent Remote Sensing” No. 41375026.

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[2] Ni Yong-nian, Cai Ying-jun, “Simultaneous synchronous spectrofluorimetric determination of vitamin B1, B2 and B6 by PARAFAC”, Spetroscopy and Spectral Analysis, Nanchang, vol. 25, pp. 1641-1644, October 2005

[3] CA Stedmon, S Markager, R Bro, “Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy”. Mar. Chem., vol. 82, pp. 239–254. March 2003

[4] KR Murphy，A Hambly，S Singh，RK Henderson，A Baker, A.; Stuetz, etc. “Organic matter fluorescence in municipal water recycling schemes: Toward a unified PARAFAC model”. Environmental Science & Technology. vol. 45, pp. 2909–2916. May 2011

[5] Stedmon, C.A.; Bro, R. “Characterizing dissolved organic matter fluorescence with parallel factor analysis: A tutorial”. Limnol. Oceanogr. Methods, vol. 6, pp. 572–579. June 2008

[6] Li, W.-H.; Sheng, G.-P.; Liu, X.-W.; Yu, H.-Q. “Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor”. Water Res. vol. 42, pp. 3173–3181. December 2008

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intrinsic fluorescence spectra characteristics of riboflavin and … · yang hui et al: intrinsic...

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