Bioorganic & Medicinal Chemistry Letters 21 (2011) 3798–3804

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis and spectroscopic characterizationof 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide as a new fluorescent probefor the determination of proteins

Yang Sun a,�, Song Wei a,�, Chen Yin a, Lusha Liu a, Chunmei Hu a, Yingyong Zhao b, Yanxi Ye c, Xiaoyun Hu c,⇑,Jun Fan a,⇑a School of Chemical Engineering, Northwest University, No. 229 Taibai North Road, Xi’an, Shaanxi 710069, Chinab Biomedicine Key Laboratory of Shaanxi Province, Northwest University, No. 229 Taibai North Road, Xi’an, Shaanxi 710069, Chinac Department of Physics, Northwest University, No. 229 Taibai North Road, Xi’an, Shaanxi 710069, China

a r t i c l e i n f o

Article history:Received 16 December 2010Revised 15 March 2011Accepted 8 April 2011Available online 19 April 2011

Keywords:1,8-NaphthalimideBovine serum albuminFluorescence spectroscopyQuantitative assay

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.04.026

⇑ Corresponding authors. Tel.: +86 29 88305252.E-mail addresses: hxy3275@nwu.edu.cn (X. Hu), fa

� Yang Sun and Song Wei are co-first authors.

a b s t r a c t

A novel 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide (BON) was synthesized as a fluorescent probefor the determination of proteins. The interactions between BON and bovine serum albumin (BSA) werestudied by fluorescence spectroscopy and UV–vis absorption spectroscopy. Fluorescence data revealedthat the fluorescence quenching of BSA by BON was likely the result of the formation of the BON–BSAcomplex. According to the modified Stern–Volmer equation, the binding constants of BON with BSA atfour different temperatures were obtained. The thermodynamic parameters, enthalpy change (DH) andentropy change (DS) for the reaction were calculated to be �23.27 kJ mol�1 and 10.40 J mol�1 K�1 accord-ing to van’t Hoff equation, indicating that the hydrogen bonds and hydrophobic interactions played adominant role in the binding of BON to BSA. Furthermore, displacement experiments using warfarin indi-cated that BON could bind to site I of BSA. The effect of BON on the conformation of BSA was also analyzedby synchronous fluorescence and three-dimensional fluorescence spectra. A new fluorescence quenchingassay of the proteins BSA using BON in the HCl–Tris (pH 7.4) buffer solution was developed with maxi-mum excitation and emission wavelengths of 373 and 489 nm, respectively. The linear range was 0.1–10.0 � 10�5 mol L�1 with detection limits were determined to be 1.76 � 10�8 mol L�1. The effect of metalcations on the fluorescence spectra of BON in ethanol was also investigated. Determination of protein inhuman serum by this method gave results which were very close to those obtained by using CoomassieBrilliant Blue G-250 colorimetry.

� 2011 Elsevier Ltd. All rights reserved.

As we know that protein are fundamental elements of life, andthe increase or decrease of protein contents in serum can displaythe conditions of human health.1 So quantitative determination ofproteins is significant in many fields, life sciences, clinical medicine,and biochemistry.2 The traditional spectrophotometry for thedetermination of proteins using dyes as analytical reagents, suchas the Lowry,3 Kjeldahl method,4 Biuret method,5 coomassie bril-liant blue,6 Amaranth,7 bromophenol blue,8 rare earth chelatesprobes,9 inorganic ions,10 indigo carmine,11 and quercetin meth-ods,12 suffer from low sensitivity, time-consuming or complicateoperations.13 In addition, the fluorescence emitted from the nativeprotein is weak14 for the detection of proteins at low concentration.To overcome these limitations, the fluorimetric methods15–17 usingcovalent and noncovalent probes not only possess high sensitivity

All rights reserved.

njun@nwu.edu.cn (J. Fan).

and selectivity, but also provide more information for the investiga-tion and determination of proteins, and the advances in their appli-cations are summarized in some reviews.18–20 Furthermore variousextrinsic dyes can be covalently attached to proteins, for example,via the e-amino group of lysine, the a-amino group of the N-termi-nus, or the thiol group of cysteine. More interesting for the analysisof pharmaceutical formulations are extrinsic dyes that interactnoncovalently with proteins and protein degradation products,for example, via hydrophobic or electrostatic interactions.21

The 1,8-naphthalimide derivatives are a special class of envi-ronmentally sensitive fluorophores and luminophore compoundsthat are widely used in various fields of science and technology.22

Because of their strong fluorescence and good photostability, the1,8-naphthalimide derivatives enjoy application in a number ofareas including the coloration of polymers,22,23 laser active med-ia,24 potential photosensitive biologically units,25 fluorescentmarkers in biology,26 analgesics in medicine,27 light emittingdiodes,28 photoinduced electron transfer sensors,29 fluorescence

300 320 340 360 380 4000

100

200

300

400

500

Flou

resc

ence

Int

ensi

ty (

a.u.

)

Wavelength (nm)

A

L

Figure 1. Fluoresence spectra of BON–BSA system (T = 293 K, kex = 280 nm).c(BSA) = 1.0 � 10�6 mol L�1; c(BON)/(10�5 mol L�1), (A–L): 0, 0.16, 0.33, 0.49, 0.64,0.81, 0.96, 1.11, 1.26, 1.41, 1.56, 1.71, pH 7.4.

0.0 0.3 0.6 0.9 1.2 1.5 1.8

1.00

1.04

1.08

1.12

1.16

293K, F0/F= 1.01+9.40*103 D

298K, F0/F= 1.01+9.03*103 D

303K, F0/F= 1.01+8.76*103 D

308K, F0/F= 1.00+8.54*103 D

F0/F

D 10-5 mol L-1

Figure 2. Stern–Volmer plots for the BON–BSA system at four different tempera-tures, pH 7.4.

Table 1Stern–Volmer quenching constants of the system of BON–BSA

T (K) KSV

( � 103 L mol�1)Kq

( � 1011 L mol�1 s�1)Ra SDb

293 9.40 9.40 0.9966 0.005298 9.03 9.03 0.9984 0.003303 8.76 8.76 0.9983 0.003308 8.54 8.54 0.9956 0.005

a R is the correlation coefficient.b SD is the standard deviation for the KSV values.

Y. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3798–3804 3799

switchers,30 electroluminescent materials,31 liquid crystal dis-plays,32 and ion probes.33

In this work, the synthesis and photophysical properties of achemosensor based on 4-butoxyethoxy-N-octadecyl-1,8-naphthal-imide (BON) (Scheme 1) were presented. The interactions betweenBON and BSA were studied by fluorescence spectroscopy. The bind-ing constants, binding sties and main sorts of binding force wereinvestigated. A novel method for determination of protein usingBON as fluorescence probe was constructed and successfully ap-plied to the determination of the total protein in human serumalbumin samples.

In this Letter, the fluorescence quenching spectra of BSA in thepresence of varying concentrations of BON were measured at 293 Kto elucidate the quenching mechanism. As seen from Figure 1 thatthe fluorescence intensity of BSA was decreased regularly with theincreasing concentration of BON, along with a small blue shift,deducing that BON could interact with BSA and quench and a com-plex was possibly formed between BON and BSA.35

Fluorescence quenching proceeds via different mechanismswhich are usually classified as dynamic quenching and staticquenching or static and dynamic quenching participate in simulta-neously. Dynamic quenching depends upon diffusion effects, hencethe diffusion coefficients and bimolecular quenching constantswould be lager with higher temperatures. While static quenchinggenerates the non-fluorescing complex which decreases the stabil-ity and values of static quenching constants with increasing tem-perature.36 The procedure of fluorescence quenching of BSAinitiated by BON could be described by Stern–Volmer equation37:

F0=F ¼ 1þ KSV½D� ¼ 1þ Kqs0½D� ð1Þ

where F0 and F denote the fluorescence intensities in the absenceand presence of quencher (BON), respectively, KSV is the Stern–Volmer quenching constant and [D] is the BON concentration, Kq

is the biomolecule quenching rate constant and Kq = KSV/s0. s0 isthe average lifetime of the molecule without any quencher andthe fluorescence lifetime of the biopolymer is 10�8 s.38 TheStern–Volmer plots of F0/F versus [D] at the four temperatures wereshowed in Figure 2, and the calculated KSV and Kq values werepresented in Table 1. The values of quenching constant KSV wasdecreased with creasing temperature and the values of Kq weremuch larger than the maximum scattering collision quenchingconstant (2 � 1010 L mol�1 s�1), indicating that the fluorescencequenching of BSA initiated by BON was static quenching.39

According to Sharma,40 dynamic quenching affected the excitedstates of molecules rather than the absorption spectra of fluores-cent substance. As seen from Figure 3 that the absorbance of BSAwas increased with the addition of BON, deducing that the quench-ing was mainly a static quenching process and at least a BON–BSAcomplex might be formed.

N OO

OO

Scheme 1. Chemical structure of 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide (BON).

Table 2The binding parameters and relative thermodynamic parameters for the system ofBON–BSA

T(K)

Kb

( � 103 L mol�1)n Ra DH

(kJ mol�1)DS(J mol�1 K�1)

DG(kJ mol�1)

293 3.89 0.93 0.9982 �23.27 10.40 �26.32298 2.82 0.90 0.9991 �26.37303 2.12 0.87 0.9995 �26.42308 1.77 0.85 0.9976 �26.47

a R is the correlation coefficient.

320 360 4000

100

200

300

BON

BON-BSA

Wavelength (nm) F

luor

esce

nce

Inte

nsity

(a.

u.)

Warfarin

Figure 5. Fluorescence spectra of BON–BSA system upon gradual addition ofwarfarin. (T = 298 K, kex = 280 nm). c(BSA) = 1.0 � 10�6 mol L�1; c(BON) =1.0 � 10�5 mol L�1; c(warfarin)/(10�6 mol L�1): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7.

200 250 300 350 4000.0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

Abs

orba

nce

H

A

Figure 3. UV–vis absorption spectra of BSA in the presence of various concentrationsof BON at room temperature. c(BSA) = 1.0 � 10–6 mol L–1; c(BON)/(10–5 mol L–1),A–G: 0, 0.16, 0.33, 0.49, 0.64, 0.81, 0.96, pH 7.4.

3800 Y. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3798–3804

When small molecules bind independently to a set of equiva-lent sites on a macromolecule, the binding constant (Kb) and num-bers of binding sites (n) can be determined by the followingequation41:

log½ðF0 � FÞ=F� ¼ log Kb þ n log½D� ð2Þ

where Kb is the binding constant for interaction of BON–BSA, n isthe number of binding sites per BSA, and F0, F, [D] have the samemeanings as in Eq. 1. The values of Kb and n could be measured fromthe intercept and slope by plotting log[(F0 � F)/F] against log[D](intercept = log Kb, slope = n) (Fig. 4), and the corresponding valuesof Kb and n were listed in Table 2. The decreasing trend of Kb withincreasing temperature was in accordance with KSV’s dependenceon temperature as mentioned above, indicating that the binding be-tween BON and BSA was moderate and a reversible BON–BSA com-plex might be formed.42 The values of n approximately are equaledto 1, indicating that there was one class of binding sites for BON inBSA. Hence, BON most likely bound to the hydrophobic pocket lo-cated in subdomain IIA.43

Binding site between BON and BSA in the presence of site Imarker (warfarin) was measured using the fluorescence titrationmethods. The concentrations of BON and BSA were stabilized atmolar ratio 10:1. Warfarin was then gradually added to theBON–BSA mixtures. An excitation wavelength of 280 nm was se-lected and the fluorescence spectra were recorded in the range of290–400 nm.44 As shown in Figure 5, with the addition of warfarin,the fluorescence decrease (about 42%) of BON bound to BSA, indi-cating the reduction in the BON binding capacity at the primarybinding site of BSA. It was very likely that BON binding occured

-5.8 -5.6 -5.4 -5.2 -5.0 -4.8

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

log

F0/ F

-1

308 K log F0/F-1 =0.85log D +3.25

303 K log F0/F-1 =0.87log D +3.33

298 K log F0/F-1 =0.90log D +3.45

293 K log F0/F-1 =0.83log D +3.59

log D mol L-1

Figure 4. Modified Stern–Volmer plots for the BON–BSA system at the fourdifferent temperatures, pH 7.4.

at the Trp 214 proximity, located in subdomain IIA of the albuminstructure (the warfarin binding pocket).45

The acting forces between a small organic molecular and bio-molecule may include hydrophobic force, electrostatic interac-tions, van der Waals interactions, hydrogen bonds. It is assumedthat the interaction enthalpy change (DH) does not vary signifi-cantly over the temperature range studied, then the thermody-namic parameters can be estimated from the van’t Hoff equation:

ln K ¼ �DHRTþ DS

Rð3Þ

The free energy change (DG) is determined from the followingrelationship:

DG ¼ DH � TDS ð4Þ

In Eq. 3, K is analogous to the binding constant Kb. The enthalpychange (DH) and entropy change (DS) were calculated from theslope and ordinate of the van’t Hoff relationship (Fig. 6), and thecorresponding results were listed in Table 2. The negative DGmeant that the binding process was spontaneous. The negativeDH and positive DS values indicated that the hydrogen bondsand hydrophobic interactions played a dominant role in the inter-actions between BON and BSA.46

The synchronous fluorescence spectra can give informationabout the molecular environment in the vicinity of the chromo-sphere molecules. When the wavelength interval (Dk) betweenexcitation wavelength and emission wavelength is 15 and 60 nm,the synchronous fluorescence spectra offer the characteristics oftyrosine and the tryptophan residues of BSA.47 Thus, the synchro-nous fluorescence spectra of Dk = 15 and 60 nm were studiedand shown in Figure 7. As seen from Figure 7(B) that the maximumemission wavelength of tryptophan residues had a slight blue shift(from 276 to 273 nm). In contrast, no obvious wavelength shift oftyrosine residues was observed (Fig. 7(A)), suggesting that thepolarity around the tryptophan residues was decreased and thehydrophobicity was increased,48 and the micro-environment

Table 3Three-dimensional fluorescence spectrum characteristics of BSA and BON–BSAsystem

Systems Parameters Peak 1 Peak 2

BSA Peak position (kex/kem) (nm/nm) 280/340 225/340Fluorescence intensity 457.7 281.4Stokes shift Dk (nm) 60 115

BON–BSA Peak position (kex/kem) (nm/nm) 280/335 225/335Fluorescence intensity 332.5 176.6Stokes shift Dk (nm) 55 110

200 250 300 350 400 450 500 5500

1000

2000

3000

4000

5000

Wavelength (nm)Fl

oure

scen

ce I

nten

sity

(a.

u.)

0.0

0.5

1.0

1.5

Abs

orba

nce

AF

Figure 9. Normalized absorption (A) and fluorescence (F) spectra of BON in ethanol.c(BON) = 1.5 � 10–5 mol L–1, (T = 293K, kex = 373 nm).

3.24 3.27 3.30 3.33 3.36 3.39 3.42

10.3

10.4

10.5

10.6

10.7

10.8 lnK = 1.25+2798.50/T; R = 0.9909ln

K

1000/T ( K-1)

Figure 6. Van’t Hoff plot for the interaction of BON and BSA, pH 7.4.

Y. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3798–3804 3801

around the tyrosine residues had no discernable change during thebinding process.

Three-dimensional fluorescence spectroscopy can comprehen-sively exhibit the information of conformational change of protein.The maximum emission wavelength and fluorescence intensity ofthe residues have a close relation to the polarity of their micro-envi-ronment. The three-dimensional fluorescence spectra of BSA andBON–BSA complex are shown inFigure 8; the corresponding param-eters are listed in Table 3. As shown in Figure 8, peak a was the Ray-leigh scattering peak (kex = kem),49 and the fluorescence intensity ofpeak a was increased with the addition of BON, deducing that a

200240

280320

280320

360400

0

100

200

300

400

ytisnetnIecnecseroul

F(

.u.a)

λem

(nm)λ

ex (nm)

a peak 2

peak 1A B

Figure 8. The three-dimensional fluorescence spectra of BSA (A) and the BON–BSA systec(BON) = 1.71 � 10�5 mol L�1, pH 7.4, T = 293 K.

250 260 270 280 290 300 310

0

20

40

60

80

100

120

140A

Wavelength (nm)

Fluo

resc

ence

Int

ensi

ty (

a.u.

)

A

L

Figure 7. Synchronous fluorescence spectra of BSA: (A) Dk = 15 nm; (B) Dk = 60 nm; c(B0.96, 1.11, 1.26, 1.41, 1.56, 1.71, pH 7.4, T = 293 K.

BON–BSA complex might come into being, which in turn resultedin the scattering effect enhanced.50 Peak 1 (kex = 280 nm,

200240

280320

280320

360400

0

100

200

300

400

λem

(nm)λ

ex (nm)

ytisnetnIecnecseroul

F(

.u.a)

peak 1

peak 2

a

m (B). (A): c(BSA) = 1.0 � 10�6 mol L�1, c(BON) = 0; (B): c(BSA) = 1.0 � 10�6 mol L�1;

250 260 270 280 290 300 3100

50

100

150

200

250

300

350 A

L

Fluo

resc

ence

Int

ensi

ty (

a.u.

)

Wavelength (nm)

B

SA) = 1.0 � 10�6 mol L�1; c(BON)/(10�5 mol L�1), A–L: 0, 0.16, 0.33, 0.49, 0.64, 0.81,

able 6ffect of interfering substances

Interference Concentration (10�6 mol L�1) Error (%)

Na+ 1.0 �0.71K+ 1.0 +0.72Ca2+ 1.0 �1.44Cu2+ 1.0 �12.46Fe3+ 1.0 �14.19Mo6+ 1.0 �10.24Zn2+ 1.0 �4.48Pb2+ 1.0 �3.10Mg2+ 1.0 �3.53Ni2+ 1.0 �4.31Cr3+ 1.0 �9.89Mn2+ 1.0 �2.66Br� 1.0 +1.60Cl� 1.0 +1.01SO4

2þ 1.0 +1.04

PO42þ 1.0 +1.52

fsDNA 6.0 +3.22ctDNA 4.0 +4.34Lysine 25.0 +3.85Tryptophane 25.0 +6.89Tyrosine 25.0 +4.41Glycine 25.0 +4.66Proline 25.0 +4.58

Figure 10. 3D–contour plot of BON in ethanol. c(BON) = 1.5 � 10–5 mol L–1,T = 293K.

400 420 440 460 480 500 520 5400

1000

2000

3000

4000

5000

0 2 4 6 8 100

500

1000

1500

2000

Wavelength (nm)

A

L

Flou

resc

ence

Int

ensi

ty (

a.u.

)

10-5

C mol L-1

F (

Flo

ures

cenc

e In

tens

ity (

a.u.

) ) F = -26.01+219.01C R=0.9992

Figure 11. Fluoresence spectra of BON–BSA system (T = 293 K, kex = 373 nm).c(BON) = 1.5 � 10�5 mol L�1; c(BSA)/(10�5 mol L�1), (A–L): 0, 0.1, 1.0, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10.0, pH 7.4.

Table 4Analytical parameters of fluorimetric method

Albumins Linear range(10�5 mol L�1)

Linearregressionequation(10�5 mol L�1)

Ra Limit ofdetection (3r,10�8 mol L�1)

SDb

BSA 0.1–10.0 DF = �26.01+ 219.01C

0.9992 1.76 0.281

a R is the correlation coefficient.b SD is the standard deviation.

Table 5Comparison of different methods for the determination of BSA

Fluorescence reagents Protein Linear range(10�5 mol L�1)

Ref.

Oxolinic acid aggregates BSA 0.1–12.1 31Hydroxypropyl-b-cyclodextrin BSA 7.6–48.5 321-Benzoyl-4-p-chlorophenylThiosemicarbazide BSA 8.1–101.5 33Organic nanoparticle biosensor BSA 0.3–5.3 34Tb3+–benzoylacetone–sodium dodecyl

benzene sulfonateBSA 0.2–9.0 35

This method BSA 0.1–10.0

400 420 440 460 480 500 520 5400

1000

2000

3000

4000

5000 blank

Zn2+

Pb2+

Ni2+

Mo2+

Mg2+

Fe3+

Cu2+

Cr2+

Ag+

Mn2+

Fluo

resc

ence

Int

ensi

ty (

a.u.

)

Wavelength (nm)

Figure 12. Fluorescence spectra of BON (kex = 373 nm) in the presence of differentmetal cations. c(BON) = 1.5 � 10�5 mol L�1; c(metal cations) = 1.0 � 10�5 mol L�1.

3802 Y. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3798–3804

kem = 340 nm) reveals the spectral characters of tyrosine and trypto-phan residues.51 The fluorescence intensity of peak 1 was decreasedby 25.16% in the presence of BON, indicating that the polarity ofboth residues decreased and more amino residues of BSA were bur-ied in the hydrophobic pocket and the binding position betweenBON and BSA might locate in this hydrophobic pocket. Accordingto literature,52 peak 2 (kex = 225 nm, kem = 340 nm) exhibits thefluorescence spectra behavior of polypeptide backbone structuresof p–p⁄ of BSA’s characteristic polypeptide backbone structure

TE

C@O. Upon addition of BON, the fluorescence intensity of peak 2 de-creased by 37.24%. The above phenomena and the analysis of thefluorescence characteristic of the two peaks in combination withthe synchronous fluorescence spectra results revealed that theinteractions between BON and BSA induced the slight unfolding ofthe polypeptides of protein, which resulted in a conformational

Table 7The determination results of total protein in human serum samples (n = 5)

Samples Sex Age G-250 method Present method Recovery (%)

mg L�1 SDa mg L�1 SDa

Human serum 1 Male 24 82.92 2.312 81.13 2.578 97.84Human serum 2 Male 42 89.63 1.119 88.92 1.635 99.21Human serum 3 Female 23 66.26 2.707 64.75 2.347 97.72Human serum 4 Female 65 69.17 2.223 67.29 2.429 97.28

a SD is the standard deviation.

Y. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3798–3804 3803

change of the protein to increase the exposure of some hydrophobicregions which were previously buried.53

It is well known that the photophysical properties of the 1,8-naphthalimides depend basically on the polarization of naphthali-mide molecule. Due to the presence of electron-withdrawing car-bonyl groups conjugated with the electron-donating substituentat C-4 position of the 1,8-naphthalimide ring, the long wavelengthband in the absorption spectra has a charge transfer character.54

Figure 9 showed an example of the absorption and fluorescencespectra of the monomeric dye in ethanol solution as an example.Figure 10 showed the three-dimensional fluorescence spectra ofBON.

The fluorescence quenching spectra of BON with varying con-centrations of BSA are shown in Figure 11. When a fixed concentra-tion of BON was titrated with different amounts of BSA, aremarkable fluorescence decrease of BON was observed, based onwhich, the working curve for detecting BSA was obtained at293 K and shown in Table 4. In comparison with other lumines-cence methods (shown in Table 5), the proposed method devel-oped in the present work had merits of wider linear range foralbumins.

The effect of 20 lL interfering substances with different concen-trations on the fluorescence intensity of BON–BSA system wastested. The fluorescence spectra were collected with the excitationand emission wavelengths of 373 and 390–550 nm. As shown in Ta-ble 6, the water-soluble amino acids and anion did not interfere oronly interfered slightly under the permission of ±5.0% relative error,whereas Fe3+, Cu2+, Mo2+ and Cr2+ produced obvious interference.

The effect of metal cations on the fluorescence spectra of BON inethanol was investigated and presented in Figure 12. As seen fromFigure 12 that the fluorescence intensity of BON was decreasedwith the addition of metal cations, which was different for everymetal cation. On the other hand, a bathochromic shift of the fluo-rescence maxima was observed. These changes could be explainedby the formation of a complex between the metal cations andBON.55 In the case of BON, the oxygen at the 1,8-naphthalimideC-4 position was action as an fluorophore donating moiety,whereas the oxygen in the butoxy fragment functions as a receptorunit. The electron-donating ability was strengthens by the interac-tions between metal cations and BON, which resulted in a red shiftin the emission. This was very different from the naphthalimidefluoroionophores with one electron-donating group, whichshowed the fluorescence enhancement through the inhibition ofPET in the presence of transition metal ions and the weakness ofthe quenching between cation and the electron-deficient fluoro-phore.56 The fluorescence intensity of BON was decreased in thepresence of metal cations, thus, the interactions between BONand BSA were further weaken.

The total protein in human serum samples (diluted about 1000times), which was got from the Fourth Military Medical University,was determined with our proposed method. The results of the ac-tual content of protein in the samples are listed in Table 7. As com-pared those from the Coomassie Brilliant Blue G-250 method,8 theaccuracy, precision, and stability of the fluorescence method aresatisfactory.

In this work, a novel 4-butoxyethoxy-N-octadecyl-1,8-naph-thalimide (BON) has been synthesized as a spectrofluorimetricprobe for the determination of proteins. The interactions betweenBON and BSA were investigated by spectroscopic methods includ-ing fluorescence spectroscopy and UV–vis absorption spectros-copy. A new fluorimetric method for the determination ofprotein has been established. Under the optimum conditions,BON could be adopted as fluorescence probes for the determina-tion of BSA. The detection limits reached the level of 10�9 mol L�1.This method was satisfactorily utilized in actual sample determi-nation. These experimental data might be available for analyticalapplication of BON as biological stain.

Acknowledgments

This study was supported in part from the National ScientificFoundation of China (No. 81001622) and Program of Xi’anIndustrial Application Technology Research and Development(CXY08007).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmcl.2011.04.026.

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