Dimethyl Sulfoxide-Free and Water-Soluble Fluorescent Probe for Detection of Bovine Serum Albumin Prepared by Ionic Co-assembly of Amphiphiles

Qin Tong, Weichun Wu, Jianghong Hu, Junhao Wang, Ke Li, Bin Dong, and Bo Song*

Article

Dimethyl Sulfoxide-Free and Water-Soluble Fluorescent Probe for

Detection of Bovine Serum Albumin Prepared by Ionic Co-assembly

of Amphiphiles

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sı Supporting Information

ABSTRACT: Detection of bovine serum albumin (BSA) is an

important issue in the sense of medical applications and enzymatic

reactions; however, the recently developed ﬂuorescent probes

require the involvement of dimethyl sulfoxide (DMSO), which

may be detrimental to proteins. In this study, we demonstrated a

DMSO-free and water-soluble ﬂuorescent probe prepared by ionic

co-assembly of amphiphiles. The cationic amphiphile is a newly

designed molecule (denoted by DPP-12) bearing a conjugated

diketopyrrolopyrrole (DPP) and two tetraphenylethylene groups.

It turns out that the ﬂuorescence emission of DPP-12 depends on

the amount of anionic amphiphilic sodium dodecyl benzene

sulfonate (SDBS). The ﬂuorescence intensity ﬁrst increases and

then decreases with the concentration of SDBS, and each branch

presents a linear relationship. BSA consumes SDBS by the formation of complexes, thus leading to an increase of ﬂuorescence

intensity of the mixed solution of DPP-12 and SDBS. Therefore, the mixed solution of DPP-12 and SDBS was applied as a

ﬂuorescent probe to detect the low concentration of BSA by back-titration. This ﬂuorescent probe does not require DMSO and has

good tolerance to metal ions in blood and good photostability. The limit of detection is as low as 940 nM, almost 3 orders of

magnitude lower than the content in organisms.

INTRODUCTION

of BSA, using a biosensing tool with simple design and high

precision, is very desirable and presently under active

■

Serum albumin is essential for maintaining the normal

nutritional status of the human body and the osmotic pressure

34−36

research.

In this regard, ﬂuorescence spectroscopy with

−8

a highly selective and sensitive probe would be potentially

applicable and prospective.

of plasma and other colloids. The content of serum albumin

in blood is an indicator of the nutritional status of an

,9−12

Diketopyrrolopyrrole (DPP) derivatives have been widely

organism.

Too much or too little cycling serum albumin

37−39

used as ﬂuorescent probes

for the detection of BSA

in the blood may be harmful to health. Too much serum

albumin may cause heart or kidney diseases,

serum albumin may cause edema or shock.

medical applications and enzymatic reactions, developing an

accurate, fast, and sensitive detection method for serum

3−15

because of their ease of synthesis, facility of modiﬁcation,

and too little

6−18

excellent durability, and attractive optical properties.

In the sense of

However, the planar aromatic DPP is water-insoluble, but

BSA is water-soluble. The contradictory solubility makes the

direct application of DPP in aqueous medium diﬃcult. In

9−23

albumin is highly demanded.

order to solve this problem, amphiphiles bearing DPP moieties

Among various serum albumins, bovine serum albumin

BSA) has structural homology with human serum albumin

43,44

were designed and synthesized.

This idea indeed works in

(

aqueous solution, but the unpredictable sedimentation

occurring between the amphiphile and BSA causes the

4−26

HSA),

is readily accessible, and thus being widely

7−30

studied as a model protein instead of HSA.

Currently, the

inaccuracy of the detection results. Therefore, a certain

developed detection approaches for BSA include Kjeldahl

method, biuret method, electrochemical impedance spectros-

copy, near-infrared diﬀuse reﬂectance spectroscopy, capillary

Received: January 9, 2021

Revised: March 28, 2021

Published: April 7, 2021

31−33

electrophoresis, and light scattering technique.

Although

these approaches show reasonable sensitivity and selectivity,

they have several disadvantages, such as the complexity of the

procedure, time consumption, and poor reproducibility.

Therefore, quick and accurate detection of minimal amounts

2021 American Chemical Society

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amount of dimethyl sulfoxide (DMSO) was usually added into

vacuum. Fourier transform infrared (FTIR) spectra were recorded on

Nicolet 6700 produced by Thermo Scientiﬁc (USA).

the aqueous solution during the sample preparation. It is

known that DMSO may cause precipitation, crystallization,

Synthesis and Characterization. Synthesis of 3,6-Bis(4-

bromophenyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (Compound

a). 1.73 g of 4-bromobenzonitrile (9.50 mmol) and 2.09 g of sodium

tert-pentoxide (19 mmol) were added into a 100 mL three-necked

ﬂask. Under nitrogen atmosphere, 12 mL of tert-amyl alcohol was

injected into the ﬂask by a syringe, and then the temperature of the

system was raised to 100 °C. After 1 h, 5 mL of tert-amyl alcohol with

47−50

and denaturation of proteins,

which will inevitably aﬀect

the detection results. Using DMSO to depress sedimentation is

not an optimal solution.

In this work, we designed and synthesized a cationic bola-

amphiphile bearing conjugated DPP and tetraphenylethylene

0.77 mL of diisopropyl succinate (3.80 mmol) was injected, and the

(

TPE) moieties in the middle of molecules (denoted by DPP-

2). It has been reported that the aromatic conjugates act as

ﬂuorescence dyes with an aggregation-induced emission

mixture was reﬂuxed at 115 °C for 24 h. Then, 5 mL of acetic acid was

added, and the mixture reacted at 120 °C for 1 h. The precipitate was

obtained by ﬁltration, and the ﬁltered cake was alternatively washed

with water (60 °C) and methanol three times. A red solid compound

a (1.23 g) was obtained with a yield of 75%.

Synthesis of Compound b. 1.02 g of compound a (2.30 mmol)

and 1.32 g of potassium tert-butoxide (11.20 mmol) were added into

a 250 mL three-necked ﬂask. Under nitrogen atmosphere, 40 mL of

dry NMP was injected, and the reactant was stirred at 60 °C for 1 h.

Then, 4.52 g of 1,12-dibromododecane (13.80 mmol) was added

dropwise. The mixture was stirred at 60 °C for 24 h. After being

cooled down, the reactant was dissolved in toluene and extracted. The

organic layer was collected and dried with anhydrous magnesium

sulfate. The product was puriﬁed by silica gel column chromatog-

raphy, using petroleum ether/ethyl acetate (10/1, v/v) as an eluent.

1−54

property,

and thus the resulting compound should have

strong ﬂuorescence emission upon self-assembly in aqueous

solution. We found that the emission of DPP-12 ﬁrst increased

and then decreased with the addition of an amount of sodium

dodecyl benzene sulfonate (SDBS), and both the increase and

decrease processes presented a linear correlation in a certain

range of concentration of SDBS. BSA and SDBS can form a

strong complex, and the reduced SDBS can be reﬂected by the

ﬂuorescence intensity of DPP-12. Therefore, DPP-12 was

applied as an indicator of back titration for the detection of

BSA. Noteworthily, organic solvents (including DMSO) were

not involved all through this study. We believe that this

research thus provides a feasible avenue for exploring DMSO-

free and water-soluble biosensors for the detection of BSA.

The reddish compound b (0.62 g) was obtained with a yield of 60%.

H NMR (400 MHz, CDCl ): δ 7.76−7.63 (m, 8H), 3.78−3.65 (m,

4H), 3.40 (t, J = 6.9 Hz, 4H), 1.85 (p, J = 6.9 Hz, 4H), 1.41 (dd, J =

.8, 6.1 Hz, 4H), 1.29−1.15 (m, 32H).

Synthesis of Compound c. 1.04 g of 1-bromo-1,2,2-triphenyl-

MATERIALS AND METHODS

■

ethylene (3.10 mmol), 1.18 g of bis(pinacol)diboron (4.65 mmol),

and 1.22 g of potassium acetate (12.40 mmol) were added into a 100

mL three-necked ﬂask. Under nitrogen atmosphere, 0.26 g of 1′-

bis(diphenylphosphine)ferrocene palladium(II) chloride (0.31 mmol)

was added, and then 20 mL of dioxane was injected with a syringe.

The mixture reacted at 80 °C for 24 h and then dissolved in DCM

and extracted with water. The organic layer was collected and dried

with anhydrous magnesium sulfate. The product was puriﬁed by silica

gel column chromatography, using petroleum ether/DCM (3/1, v/v)

Materials. Sodium tert-pentoxide, 1,12-dibromododecane, chloro-

form-d (CDCl ), and DMSO-d were purchased from J&K Chemical

Co., Ltd. (Shanghai). Bis(pinacol) diboron and Pd(PPh₃)₄were

purchased from Energy Chemical Co., Ltd. Tetrahydrofuran (THF),

N-methyl pyrrolidone (NMP), methanol, pyridine, Na SO , NaCl,

K CO , KCl, CaCl , MgSO , and ZnCl were bought from Sinopharm

Chemical Reagent Co., Ltd. (Shanghai). SDBS, diisopropyl succinate,

and 1-bromo-1,2,2-triphenylethylene were purchased from TCI

Development Co., Ltd. (Shanghai). Ethyl acetate, petroleum ether,

and dichloromethane (DCM) were purchased from Yonghua

Chemical Technology Co., Ltd. (Jiangsu). 1,4-Dioxane and tert-

pentoxide were bought from 3A Chemicals Co., Ltd. (Shanghai).

Pd(dppf)Cl ·CH Cl , sodium tosylate (ST), and sodium laurate (SL)

as an eluent. A white solid compound c (0.65 g) was obtained with a

yield of 74%. H NMR (400 MHz, CDCl ): δ 7.35 (dq, J = 5.2, 2.7

Hz, 2H), 7.31 (d, J = 6.8 Hz, 3H), 7.14 (t, J = 7.1 Hz, 2H), 7.11−7.07

(m, 4H), 7.07−7.03 (m, 2H), 6.97 (dd, J = 6.5, 2.9 Hz, 2H), 1.13 (s,

12H). ESI-MS (m/z): [M + 1] calcd for C H BO , 383.210; found,

26 27

were purchased from Macklin Biochemical Technology Co., Ltd.

Shanghai). 4-Bromobenzonitrile was purchased from Titan Scientiﬁc

383.218.

(

Synthesis of Compound d. 0.28 g of compound b (0.30 mmol),

0.25 g of compound c (0.66 mmol), and 0.02 g of tetrakis-

(triphenylphosphine)palladium were added to a 100 mL three-necked

ﬂask. Under nitrogen protection, 10 mL of THF and 5 mL of

potassium carbonate solution (2 M) were added, and the mixture

reacted at 66 °C for 24 h. The reactant was extracted with water and

DCM, and the organic layer was dried with anhydrous magnesium

sulfate. The crude product was puriﬁed by silica gel column

chromatography, using petroleum ether/DCM (1/2, v/v) as the

eluent. An orange solid compound d (0.22 g) was obtained with a

yield of 57%. H NMR (400 MHz, CDCl ): δ 7.57 (d, J = 8.3 Hz,

4H), 7.11−7.00 (m, 34H), 3.67 (t, J = 7.7 Hz, 4H), 3.40 (t, J = 6.8

Hz, 4H), 1.85 (p, J = 7.1 Hz, 4H), 1.12 (s, 36H). MALDI-TOF-MS

Co., Ltd. (Shanghai). Newborn calf serum was bought from Beyotime

Biotechnology Co., Ltd. Milli-Q water with a resistivity of 18.2 MΩ·

cm was produced by Direct-Q 5UV manufactured by Merck

Millipore.

Instruments. H NMR spectroscopy was performed on Avance III

00 MHz (Bruker, USA) and 600 MHz NMR spectrometers (Agilent

Technologies, USA). Mass spectroscopy (MS) of 2,5-bis(12-

bromododecyl)-3,6-bis(4-bromophenyl)pyrrolo[3,4-c]pyrrole-1,4-

(2H,5H)-dione (compound b) was performed on a micro Q-TOF III

mass spectrometer (Bruker, USA) using electrospray ionization (ESI)

mode, and the mass spectra of 4,4,5,5-tetramethyl-2-(1,2,2-triphe-

nylvinyl)-1,3,2-dioxaborolane (compound c), 2,5-bis(12-bromodo-

decyl)-3,6-bis(4-(1,2,2-triphenylvinyl)phenyl)pyrrolo[3,4-c]pyrrole-

(m/z): [M] calcd for C H Br N O , 1290.504; found, 1290.706.

,4(2H,5H)-dione (compound d), and DPP-12 were obtained on a

matrix-assisted laser desorption/ionization time-of-ﬂight MS

MALDI-TOF-MS, Bruker, USA) system. The ﬂuorescence spectra

Synthesis of DPP-12. 0.12 g of compound d (0.1 mmol) was added

in 100 mL of pyridine and reﬂuxed at 100 °C for 72 h. Pyridine was

removed by Rotavapor, and the solid was washed ﬁve times with ethyl

ether. A reddish solid DPP-12 (0.11 g) was obtained with a yield of

75%. At 25 °C, 0.094 g DPP-12 can be dissolved in 100 mL of water,

(

were recorded on FLS980 (Edinburgh Instruments, UK). The

ultraviolet−visible (UV−vis) absorption spectra were recorded on

Cary 60 (Agilent Technologies, USA). Atomic force microscopy

and the melting point of DPP-12 is 261 °C. H NMR (400 MHz,

(AFM) images were recorded on a MultiMode 8 microscope (Bruker,

DMSO-d ): δ 9.06 (d, J = 6.0 Hz, 4H), 8.58 (t, J = 7.8 Hz, 2H), 8.14

USA). Peak force quantitative nanomechanical mapping mode with a

(t, J = 7.0 Hz, 4H), 7.69−6.78 (m, 38H), 4.56 (t, J = 7.5 Hz, 4H),

−

ScanAasyst-air probe (nominal spring constant of 0.4 N m ,

frequency 70 kHz, from Bruker) was adopted during the measure-

ment. The samples were cast on a mica substrate and dried in a

3.60 (s, 4H), 2.03−1.92 (m, 4H), 1.22 (s, 36H). C NMR (100

MHz, DMSO-d ): δ 161.40, 147.16, 146.21, 145.48, 144.73, 142.79,

142.74, 142.53, 142.01, 139.72, 131.00, 130.69, 130.60, 128.08,

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Scheme 1. Synthetic Route of DPP-12

28.03, 127.97, 127.88, 126.86, 125.83, 108.60, 60.73, 30.72, 28.91,

8.86, 28.81, 28.77, 28.41, 28.31, 28.25, 25.92, 25.41. MALDI-TOF-

MS (m/z): [M − Br] calcd for C H Br N O , 1369.676; found,

1369.876.

RESULTS AND DISCUSSION

■

The synthetic route of DPP-12 is shown in Scheme 1, and the

details of each step of reaction are described in the Materials

and Methods section. The characterization, mainly by H

NMR and MS, of the synthesized products is presented in the

Supporting Information . DPP-12 is soluble in water, and the

aqueous solution shows an emission peak at around 595 nm

Figure 1. (a) Fluorescence spectra of DPP-12 in the presence of

diﬀerent concentrations of SDBS in aqueous solution. (b) Plot of

ﬂuorescence intensity at 595 nm vs C_S_D_B_S

(λ_e_x= 505 nm). The overlap between the absorption and

emission bands (Figure S1) is quite small, which means that

the self-absorption should be negligible. The plot of

ﬂuorescence intensity versus concentration was employed to

determine the critical micelle concentration (CMC) of DPP-

co-assembly of DPP-12 and SDBS through electrostatic

interactions. (1) As the concentration of SDBS (denoted by

−

−1

C_S_D_B_S) is lower than 1.1 × 10 mol L , SDBS cannot form

micelles alone but co-assemble with DPP-12. As the

concentration of DPP-12 adopted here is higher than its

CMC, the molecules exist in the solution in the form of

micelles instead of individual molecules. Presumably, SDBS

molecules were inserted into the micelles of DPP-12, thus

forming new assemblies by the complexes of DPP-12 and

SDBS. The electrostatic combination will, to some extent,

screen the repulsive interaction between the pyridinium groups

on DPP-12, and the aromatic conjugates of DPP and TPE that

are present inside the micelle have more chances to form

closely packed stacking. This could be a reasonable explanation

5,56

As shown in Figure S2 , the linear ﬁtting of the data

−

−1

shows an intersection point at 3.3 × 10 mol L , which is

assigned to the CMC of DPP-12. In the following study, a

concentration of 1 × 10⁻mol L (above CMC) was adopted.

We actually have attempted to use DPP-12 as the

ﬂuorescent probe to detect BSA directly but failed because

of the formation of precipitate once BSA was added into the

aqueous solution of DPP-12 (Figure S3), and the precipitation

makes the ﬂuorescent signals NOT reliable. This result is

−1

consistent with that reported in the literature. We are pleased

to ﬁnd that the ﬂuorescence emission of DPP-12 responds to

SDBS. By increasing the amount of SDBS while keeping the

concentration of DPP-12 constant, the ﬂuorescence emission

ﬁrst increases and then decreases until it maintains a constant

intensity, as shown in Figure 1. Through linear ﬁtting, we got

to the enhanced emission of DPP-12. (2) As C

is higher

SDBS

−

−1

than 1.1 × 10 mol L , SDBS tends to form micelles. The

excess SDBS micelles may encapsulate DPP-12 molecules and

thus decompose the initially formed co-assemblies, leading to

the decrease of ﬂuorescence emission. When there are enough

SDBS micelles to hold all the DPP-12 molecules separately, the

ﬂuorescence will not decrease any more, which also explains

an intersection point at 1.1 × 10⁻mol L . Coincidently, this

−1

−

concentration is very close to the CMC of SDBS (1.2 × 10

mol L ). These results can be reasonably explained by the

−

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SDBS. As shown in Figure 2 , increasing the concentration of

Article

the constant ﬂuorescence intensity at high concentrations of

SDBS. It is worth noting that no observable precipitate

appeared in the solution, no matter how much SDBS was

added.

To understand the roles played by the alkyl chains and

tosylate in the interaction with DPP-12, ST and SL were

selected as controls, and the spectral results are shown in

Figure S4. ST remarkably reduced the emission of DPP-12.

Similar to SDBS, SL ﬁrst improved and then reduced the

emission of DPP-12 by increasing the addition amount.

Diﬀerent with that occurred with SDBS, the changes of

ﬂuorescence intensity are fairly small. These results indicate

that both the tosylate (showing strong interactions with

pyridinium) and the alkyl chains (hydrophobic interactions)

are indispensable for the dramatic and parabolic change of

ﬂuorescence emission. Fortunately, it is proven later in this

study that the complexes formed by SDBS and DPP-12 also

showed very good responses to BSA.

We used the absorption spectra to monitor the aggregation

state of DPP-12 in the presence of diﬀerent concentrations of

Figure 3. AFM images of the co-assemblies of DPP-12 and SDBS

−

−3

with C

L .

= (a) 0, (b) 2 × 10 , (c) 1 × 10 , and (d) 5 × 10 mol

SDBS

−

believe that these particles should be mainly composed of

SDBS molecules with a few DPP-12 molecules in each particle.

The morphological change corresponds to the ﬂuorescence

decrease as too much SDBS was added to the solution. Based

on the above discussion, a schematic illustration of the

morphological alternation of the assemblies is summarized in

Scheme 2.

Figure 2. UV−vis spectra of DPP-12 in the presence of diﬀerent

concentrations of SDBS. The concentration of DPP-12 (C

) is 1

DPP‑12

−

−1

10 mol L .

Scheme 2. Schematic Illustration of the Morphological and

Fluorescence Emission Change of DPP-12 as Being

Interacted with SDBS and BSA

SDBS leads to the shift of the peak from 500 to 508 nm. The

bathochromic shift implies that the DPP moieties were packed

more closely in the aggregates, which should be ascribed to the

balanced electronic repulsion eﬀect between the pyridinium

head groups due to the insertion of the negatively charged

8,59

−3

−1

tosyl groups.

(

When C_S_D_B_Sincreased to 5 × 10 mol L

approximately 50 times of that of DPP-12), the absorption

peak shifted back. SDBS dominates in the assemblies, and

DPP-12, to a greater extent, is segregated by SDBS molecules.

This might be a reasonable explanation for the shift of the

absorption peak.

The abovementioned assumption that SDBS is inserted into

the assemblies of DPP-12 can be proved by the morphological

change of the assemblies. As shown by the AFM images in

Figure 3, DPP-12 formed irregular nanodiscs with thicknesses

of 2−3 nm and diameters in the range of 30−80 nm. As 2 ×

−

−1

mol L SDBS was added into the solution of DPP-12 (1

The interaction between DPP-12 and SDBS was qualita-

tively analyzed by FTIR spectroscopy. As shown in Figure 4a,

the peaks at around 1205 and 1133 cm are assigned to the

−

−1

10 mol L ), the thickness of the nanodiscs kept

unchanged, whereas the diameters increased to the range of

00−450 nm. The insertion of SDBS into the assemblies can

well explain the size increase of the nanodiscs. As C

−

antisymmetric and symmetric stretching vibrations of −SO

−

in SDBS, respectively. When SDBS was added to 2 × 10 mol

L , the two peaks shift to 1195 and 1128 cm , respectively,

which should be attributed to the electrostatic interaction

between the sulfonate and pyridinium groups. When the

concentration of SDBS increased to 1 × 10 or 5 × 10 mol

L , the signal of the complex was covered by the signal of the

SDBS

increased to 1 × 10⁻³mol L , the nanodiscs shrank to 80−

00 nm. Now, the ratio of SDBS to DPP-12 is 10:1, and SDBS

became dominating. The excess SDBS forced the nanodiscs to

−1

−

−3

break down into smaller ones. As C

increased to 5 × 10

SDBS

−

−1

mol L , 50 times of that of DPP-12, in the vision of AFM

image, only particles with size of 30−40 nm were observed. We

sulfonate groups, and hence the vibrational peaks shifted back

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between the ﬂuorescence intensity and the concentration of

Article

BSA (C_B_S_A) in the range of 0−5 μM, as shown in Figure 5b. By

linear ﬁtting the data, we can also achieve the limit of detection

(

LOD) by the equation LOD = 3σ/K, where σ refers to the

standard deviation calculated from 10 times the measurement

of the ﬂuorescence intensity at 595 nm of the blank sample,

and K is the slope of the ﬁtting line. LOD of the mixture

probe for BSA is 940 nM, which is far below the limit in

organisms (600 μM).

How could BSA improve the ﬂuorescence emission of BSA-

probe and even build a good linear relationship? The

absorption spectra can be used to answer this question. By

carefully controlling the concentration of BSA in the mixed

solution, we found that decreasing the concentration of SDBS

and increasing the amount of BSA have the same eﬀect, both

leading to the peak shift from 500 to 508 nm and both

resulting in the enhancement of absorption intensity, as shown

in Figures 2 and 6 . These results suggest that BSA should have

Figure 4. FTIR spectra of (a) SDBS and (b) DPP-12 with diﬀerent

C_S_D_B_Svalues. C_D_P_P_‑₁₂₌₁_×₁₀− mol L .

−1

−

to 1205 and 1133 cm . As shown in Figure 4b, the peaks at

711 and 1674 cm⁻are assigned to the stretching vibration of

the carbonyl and stretching vibration of CC in the DPP

moiety. Upon the addition of 2 equiv SDBS, the peak at

around 1711 cm⁻becomes stronger. This alteration could be

explained by the reduced interaction between DPP-12

molecules due to the intercalation of SDBS. The conjugation

or delocalization eﬀect produced by the strong π−π stacking

interaction suppresses the characteristic peak of the amide

carbonyl group. The segregation eﬀect caused by SDBS

intercalation will reduce the π−π stacking interaction, so the

suppressed amide carbonyl characteristic peak becomes

obvious. Because of the reduced π−π stacking interaction,

−

the vibrational peak of CC shifted to 1678 cm . These

results are consistent with the change in morphology and can

explain the cause of the ﬂuorescence change together.

Figure 6. UV−vis spectra of the mixed aqueous solution of DPP-12

and SDBS in the presence of diﬀerent concentrations of BSA. C

= 1 × 10 mol L .

DPP‑12

−

−1

In the following, the mixed solution of DPP-12 and SDBS

was applied to detect BSA, in which the ﬂuorescence intensity

of DPP-12 acts as an indicator. Through varying the

concentration of SDBS and keeping the concentration of

a speciﬁc interaction with SDBS, and the addition of BSA will

consume a deﬁned amount of SDBS in the mixed solution,

thus leading to an increase in the ﬂuorescence emission.

It has been well documented that the combination of BSA

and SDBS will result in an enhanced emission of SDBS

DPP-12 at 1 × 10⁻mol L (corresponding spectral results

−1

are shown in Figure S5 ), the optimal concentration of SDBS

−

−1

was 1.7 × 10 mol L . For convenience of expression, the

mixed solution with the above-mentioned optimal concen-

trations is denoted by BSA-probe. Under these speciﬁc

conditions, reducing SDBS leads to an increase in the

ﬂuorescence intensity of the BSA-probe, which means that

the addition of BSA will improve the ﬂuorescence emission of

the BSA-probe by reducing the concentration of SDBS. The

spectral responses of BSA-probe to diﬀerent amounts of BSA

are presented in Figure 5a. A linear relationship was built

residues at around 350 nm. This was veriﬁed in our case too.

Now, focusing on the ﬂuorescence between 300 and 500 nm,

we found that the emission centered at 350 nm indeed

increased upon the addition of BSA (Figure S6). Concom-

itantly, the ﬂuorescence peak at around 330 nm kept

unchanged. This is the result of the combination between

BSA and SDBS. They are close enough that the energy of

tryptophan and tyrosine residues on BSA is able to be

transferred to SDBS. These results were consistent with that

reported in the literature.

Next, the tolerance of BSA-probe to the metal ions in blood

was investigated. Na , K , Ca , Mg , and Zn are commonly

found in blood, and herein, the concentrations of the ions were

controlled to the maximum values. As shown in Figure 7a, the

above ions have little eﬀect on the ﬂuorescence intensity.

Based on the inﬂuence of metal ions on the ﬂuorescence

intensity, we were able to extrapolate the relative errors to the

detection of BSA. The results are listed in Table 1. The

maximum relative error was −6.89%, indicating that BSA-

probe works nicely even in the presence of these ions.

The photostability of the ﬂuorescent probe was investigated

by irradiating the solution by 505 nm light with a power of 2.5

W. As shown in Figure 7b, the ﬂuorescence intensity showed a

trivial decrease with the irradiation time. The emission

maintained 97% of the initial intensity after the samples were

Figure 5. (a) Fluorescence spectra of the mixed solution of DPP-12

and SDBS in the presence of diﬀerent concentrations of BSA. Inset:

photographs of the mixed solution of DPP-12 and SDBS in the

presence of diﬀerent concentrations of BSA. (b) Plot of ﬂuorescence

= 1.7 × 10⁻mol L . C

−1

= 1 ×

intensity at 595 nm vs BSA. C

SDBS

DPP‑12

−

−1

mol L .

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The Supporting Information is available free of charge at

Article

Figure 7. (a) Relative ﬂuorescence intensity at 595 nm in the presence of diﬀerent metal ions. (b) Photostability of DPP-12. The sample was

continuously irradiated by 505 nm light (2.5 W) for 30 min.

Table 1. Eﬀect of Foreign Metal Ions on the Determination

of BSA

The ﬂuorescent probe formed by the complex of DPP-12

and SDBS is not ﬂawless. Herein, for a fair evaluation, the

advantages and disadvantages of the probe are summarized and

listed in Table 3.

metal

ions

concentration

founded

−1

relative error

(%)

BSA

−

−1

−6

(×10 mol L

)

(×10 mol L )

Na⁺

145

5.23

5.03

5.08

4.71

4.66

4.67

0.61

1.58

−5.84

−6.89

_K+

Table 3. Advantages and Disadvantages of the Fluorescent

Probe Formed by the Complex of DPP-12 and SDBS

5.51

1.33

1.01

0.02

_C_a₂+

Mg²⁺

Zn²⁺

advantages

DMSO-free

disadvantages

poor selectivity

water-soluble

good photostability

high accuracy

narrow probing range

indirect probing

irradiated for 30 min, indicating that the probe owns good

photostability under the working conditions.

Finally, BSA-probe was practically applied to detect the BSA

content of unknown samples from newborn calf serum. In

order to evaluate the detection capability of the probe at

diﬀerent concentrations of BSA, the newborn calf serum was

diluted to diﬀerent concentrations and then added into BSA-

probe, where the concentrations of DPP-12 and SDBS were 1

CONCLUSIONS

■

−

−3

−1

10 and 1.7 × 10 mol L , respectively. C

in the

In this study, an amphiphile containing a conjugate of one

DPP and two TPE groups (i.e., DPP-12) was designed and

synthesized. The ﬂuorescence emission of DPP-12 ﬁrst

increases and then decreases with the concentration of added

SDBS. In a certain range of concentration, the ﬂuorescence

intensity of DPP-12 shows a linear relationship with the

amount of SDBS. Another linear relationship is built between

the content of BSA and emission of DPP-12 mediated by

SDBS, for BSA consumes SDBS in the mixed solution, thus

leading to the increase of ﬂuorescence emission of DPP-12.

The mixed solution working as a ﬂuorescent probe for the

detection of BSA shows very good photostability and high

accuracy.

BSA

sample after diluting was obtained by substituting the

ﬂuorescence intensity at 595 nm into the original linear ﬁtting

equation (in Figures 5b and S7a). For comparison, we also

determined the content of BSA by using UV−vis absorption

spectra (in Figure S7b ). In doing so, pure BSA was employed

to build a relationship between the absorption intensity and

the concentration of BSA, as shown in Figure S8. Then, the

concentrations of BSA in the unknown samples were read from

the plot. The results obtained from these two methods are

shown in Table 2. First, the two groups of data are comparable

Table 2. Detection of BSA of Unknown Samples

DPP-12 + SDBS

UV−vis spectra

ASSOCIATED CONTENT

sı Supporting Information

■

RSD

(%)

BSA

RSD

(%)

BSA

(×10⁻⁶mol L⁻¹

sample

(×10⁻mol L⁻¹

)

https://pubs.acs.org/doi/10.1021/acs.langmuir.1c00072.

1.52

4.54

4.99

1.98

1.34

1.22

1.59

4.62

5.11

1.38

1.33

2.31

UV−vis and ﬂuorescence spectra of DPP-12; CMC

determination of DPP-12; photographs of the solutions

of DPP-12, DPP-12 + BSA, and DPP-12 + SDBS + BSA;

ﬂuorescence spectra of DPP-12 in the presence of ST

and SL; ﬂuorescence spectra of SDBS and the mixed

solution of DPP-12 and SDBS in the presence of BSA;

content of BSA determined by UV−vis absorption

for each sample. As shown in Table 2, the BSA concentrations

determined by the UV−vis absorption method were relatively

higher that those obtained from the ﬂuorescence method. We

assume that the discrepancy may be caused by the scattering

eﬀect in the absorption spectra, and the scattering has little

eﬀect on the ﬂuorescence emission. From this point of view,

the ﬂuorescence method may provide more accurate results.

spectra; H NMR spectra of compounds b, c, d, and

DPP-12; C NMR spectrum of DPP-12; and MS of

compounds b, c, d, and DPP-12 (PDF )

537

Langmuir 2021, 37, 4532−4539

Langmuir

Article

8) Mafina, M.-K.; Hing, K. A.; Sullivan, A. C. Development of novel

AUTHOR INFORMATION

Corresponding Author

Bo Song − College of Chemistry, Chemical Engineering and

Materials Science, Soochow University, Suzhou 215123, P. R.

China; orcid.org/0000-0002-7546-9482;

Email: songbo@suda.edu.cn

■

fluorescent probes for the analysis of protein interactions under

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status. J. Parenter. Enteral Nutr. 1980, 4, 450−454.

Authors

Qin Tong − College of Chemistry, Chemical Engineering and

Materials Science, Soochow University, Suzhou 215123, P.

R. China

Weichun Wu − College of Chemistry, Chemical Engineering

and Materials Science, Soochow University, Suzhou 215123,

P. R. China

Jianghong Hu − College of Chemistry, Chemical Engineering

and Materials Science, Soochow University, Suzhou 215123,

P. R. China

Junhao Wang − College of Chemistry, Chemical Engineering

and Materials Science, Soochow University, Suzhou 215123,

P. R. China

Ke Li − College of Chemistry, Chemical Engineering and

Materials Science, Soochow University, Suzhou 215123, P.

R. China

Bin Dong − College of Chemistry, Chemical Engineering and

Materials Science, Soochow University, Suzhou 215123, P.

R. China; orcid.org/0000-0001-5681-372X

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Complete contact information is available at:

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(

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Notes

The authors declare no competing ﬁnancial interest.

(

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ACKNOWLEDGMENTS

■

The authors are thankful to the National Natural Science

Foundation of China (21674075), the Natural Science

Foundation of Jiangsu Province (BK20161211), the Key

University Science Research Project of Jiangsu Province

(

20) Lu, H.; Wang, K.; Liu, B.; Wang, M.; Huang, M.; Zhang, Y.;

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17KJA150007), Suzhou Key Laboratory of Macromolecular

Design and Precision Synthesis, Jiangsu Key Laboratory of

Advanced Functional Polymer Design and Application, a

project funded by the Priority Academic Program Develop-

ment of Jiangsu Higher Education Institutions, and State and

Local Joint Engineering Laboratory for Novel Functional

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