Fluorescence Quenching Studies of Near-Infrared Fluorophores

Fluorescence Quenching Studies of Near-Infrared Fluorophores

DANIEL ANDREWS-WILBERFORCE and GABOR PATONAY* Department of Chemistry, Georgia State University, Atlanta, Georgia 30303

Near-infrared laser dyes were studied with the use of conventional fluorescence quenching. A systematic study of the effects of different quenchers and temperature on the fluorescence of near-infrared fluorophores is presented. Differences in the quenching of near-infrared fluorophores were observed for quenching by ionic quenchers. Quenching with ionic and nonionic quenchers led to nonlinear quenching results.

Index Headings: Near IR; Fluorescence; Quenching.

INTRODUCTION

Alcohol-soluble polymel~hine dyes have the ability to form complexes in aqueous solution with organic molecules and ions. Several of the polymethine dyes have their absorbance band in the near-infrared region where recently developed laser diodes have their output maximum. Near-infrared fluorophores have not been utilized to their fullest potential, mostly because of the lack of detailed studies on near-infrared optical properties of polymethine dye complexes.

Polymethine dyes are ]known to have an absorption band in the far-visible or in the near-infrared (IR) region (550-900 nm). Some of these compounds have very large molar absorptivities and are strongly fluorescent. Con- sequently, they may be detected and identified at ultra- trace levels. 1 The use of polymethine dye complexes has recently received some attention in the area of separation science. 2,3 Protein in human serum was labeled with in- docyanine green, and the complex was separated by a gel filtration column2 The detection limit was about two orders of magnitude better than the values obtained by conventional methods. Bound functionalized and non- functionalized near-infrared dyes may also find application as labeling agents in other areas of chemical analysis, especially in biologically and clinically important determinations.

Polymethine dyes and functionalized polymethine dyes have also received attention as saturable absorbers in mode-lock laser systems. 4,5 As mode lockers, these dyes are responsible for very short laser pulses, in the pico- second range, which extend the time scale of chemical investigations down close to the period of a molecular vibration. Consequently, the photophysical properties of polymethine dyes have been of interest in several areas of investigation, s'7 Cooper and Rome have used the ex- ternal heavy atom effect to investigate the influence of the triplet state in the photoisomerism of thiacarbocy- anines. 8 The reported increase in trans to cis photoisom- erization rate is indicative of triplet state involvement. Several studies reported the existence of delayed fluorescence in cyanine dyes due to impurities? Excited states, laser properties, and stabilitiy have been investigated by

Received 1 March 1989; revision received 1 June 1989. * Author to whom correspondence should be sent.

several researchers. 1°,11 These dyes were shown to improve stability in the absence of oxygen. Increasing dye concentration was also found to improve the useful lifetime of the lasing media.

Nevertheless, the use of near-infrared laser dyes has not gained considerable attention in analytical applica- tions, in spite of the large number of such dyes that are available. The high molar absorptivity of these dyes en- hances ultrasensitive detection. When near-infrared dyes are used as labels, assays can be carried out in the long wavelength region to eliminate blank interference from various impurities. The application of near-infrared chromophores as labels is especially advantageous when laser diodes are used as excitation sources?

Fluorescence quenching extends the dimensionality of the fuorescence measurement by specifically introducing a chemical probe into the immediate vicinity of a fluorophore. When different quenchers are employed, fluorescence quenching provides analytical information on the chemical environment (e.g., the hydrophobicity and ionic nature) of the fluorophore. One must take care when applying fluorescence quenching to studies of near-infrared fluorophores. In some instances, the quencher may interact with the near-infrared fluorophore to decrease the fluorescence, resulting in the transition to the ground state by processes other than dynamic or static quenching. For example, the quencher may affect the extended conjugation of the near-infrared fluorophore, with a corresponding change in fluorescence intensity.

Ion-pair fluorescence quenching of cyanine dyes has been investigated recently. 12 The quenching of cationic polymethine dyes by anionic quenching dyes was found to be dependent on the ground-state interaction of the anionic quenching dyes with the cationic polymethine. 2,4,6-Trinitrobenzenesulfonate has been found to be an effective quencher of the fluorescence of carbocyanine dyes used for labeling of membranes. 13 The quenching was determined to occupy by complex formation rather than a collisional mechanism.

In this paper, a systematic study of the influence of quenchers on the fluorescence of near-infrared laser dyes is described. Different ionic and nonionic quenchers were used to evaluate the role of electric charge of the quencher molecules. All systems in this study were characterized by Stern-Volmer plots and constants. Information gained by the use of these quenchers contributes to the investigation and characterization of near infrared dyes. Quencher-induced perturbations are examined with fluorescence and absorbance. These data provide additional insight into the mechanism of interaction between near- infrared dyes and the chemical environment. The information obtained is important to the design of appropri- ate labeling systems. Furthermore, insight into the susceptibility of the near-infrared laser dye molecule is

1450 Volume 43, Number 8, 1 9 8 9 0003-7028/89/4308-145052.00/0 APPLIED SPECTROSCOPY © 1989 Society for Applied Spectroscopy

presented. A particular emphasis will be placed on the problems associated with commonly used ionic and nonionic quenchers.

EXPERIMENTAL

R e a g e n t s and C h e m i c a l s . The near-infrared laser dyes, IR-125, IR-140, IR-144, 8,8'-diethyldicarbocyanine iodide (DTDCI), 3 ,8 ' -die thyl t r icarbocyanine iodide (DTTCI), 1,1',3,8,3',3'-hexamethylindotricarbocyanine perchlorate (HITCP), and 1,1'3,8,8'3'-hexamethyl- 4,4'5,5'-dibenzo-2,2'-indotricarbocyanine perchlorate (HDITCP), were obtained from Eastman Kodak Com- pany (Rochester, NY) and were used as received. Stock solutions (10 -3 M) of the near-infrared laser dyes were prepared in spectrophotometric-grade methanol (Al- drich). For the quenching studies, fresh solutions were prepared by pipetting the required amount of near-infrared laser dye stock solution dissolved in methanol into a 50-mL volumetric fask. The methanol was evaporated by using dry nitrogen gas. The volumetric flask was then filled with deionized water or organic solvent and briefly sonicated. Solutions in equilibrium with air were used in this study. The chemical structures and the maximum emission and excitation wavelengths of the near-infrared dyes used in this study are listed in Table I.

The quenching probes- -n i t romethane , t r imethyl- amine, and 2-nitro-l-propanol--were purchased from Aldrich Chemical Company (Milwaukee, WI). KI and Cu(II)sulfate were obtained from Fisher Scientific Com- pany (Fair Lawn, NJ).

Ins trumentat ion . Fluorescence spectra were recorded on an SLM 8000 spectrofluorometer. The spectrofluorometer is interfaced to an IBM PS/2 computer. This combination allowed storage of the entire emission or excitation spectrum on disk in digitized form. Storage of the entire emission spectrum in digital form provides several advantages. With the use of the entire emission spectrum, the true quantum efficiency ratios may be calculated, even when the fluorophore interacts with quenchers, and the emission spectrum undergoes changes. Quantum efficiency ratios were calculated by integration of the fluorescence spectral area.

Data Analysis. The data were analyzed by the Stern- Volmer equation:

Io/I = Ksv [Q] + 1 (1)

where Ksv is the Stern-Volmer quenching constant, [Q] is the concentration of the quencher, and I0 and I are the intensity of fluorescence in the absence and presence of quencher, respectively. Traditional Stern-Volmer plots for the near-infrared laser dyes were obtained by using intensity values determined by integrating the area under the fluorescence emission curves. Only those intensity values corresponding to data points containing spectral information were included in the calculations. The fluorescence spectra were acquired with excitation wavelengths corresponding to the maximum excitation wavelength for each near-infrared laser dye. The total fluorescence has been calculated from the digitized data with the use of the integration program supplied with the spectrofluorimeter. Very small (< 1 nm) spectral shifts were observed upon addition of quenchers. This spectral

TABLE I. The chemical structures and the maximum emission and excitation wavelengths of the near-infrared dyes used in this study.

IR 125 ~t/ex ~em

,, ca, ~ - - ~ / 780 815 It'('CH = CH) ~ "~'lq ~ " ~

I I (CHz)4SO3Na (CH2)4S030

IR132 ~ N ~

~ - - ~ N , ' ~ C H - C H = [ " ~ - C H = C H " ~ ~

(~H 2)3C02CH3 (~H 2)3COzCH3 CtO40

764 824

C1040

C02C2Hs IR 144 I N

N H C CHs "~. t ' ~ ' ~ c n , ~ 698 708 "~N'~"~ CH- CIt = CH=C

[ (CH 2)3SO3 ~) (CH2)3SO 3 H "N (C2H$) 3

DTDCI

t rO i C2Hs C2Hs

D T T C I

i i C~Hs I 0 C2Hs

647 668

746 777

DODCI

C2H 5 C2Hs

577 605

DOTCI

i i C2H5 I~) C~Hs

H I T C P ~ = H 3 CH3

CH 3 CH3 - f ~ - ~ CH qCn = CHh"~,,",~

~H~ clo,O 'c~

678

736

703

764

~.~ c . ~ - t - ~ V

~"3 c~o,O 'c., 771 805

APPLIED SPECTROSCOPY 1451

TABLE II. Summary of the effect of quenchers on near-infrared dyes.

Near-infrared dye Quencher IR-125 IR-144 IR-140 IR-132 HDITCP H I T C P D T T C I DTDCI

Cu(II) Q~ Q Q NQ b Q Q Q Q Ki Q NQ NQ NQ NQ Q NQ NQ 2-nitro-l-propanol Q NQ NQ NQ NQ Q NQ NQ Nitromethane NQ NQ NQ NQ NQ NQ Q NQ Trimethylamine NQ NQ NQ NQ NQ NQ NQ Q Solvent W ~ W W Et0H (acid) W W W W

Q: Quenching was observed. b NQ: No significant quenching was observed. W: Aqueous solvent.

shift does not affect the calculation of the total peak area. Little or no absorption by the quenchers was observed at the excitation/emission wavelengths of the near- infrared laser dyes.

M e t h o d . Temperature regulation to better than 0.1°C was achieved by using a Haake A80 (F.R.G.) bath and circulator. The cell holder 'was also thermostatted at the desired temperature by using the circulator. The thermostatted cell holder was equipped with a magnetic flea stirrer to maintain homogeneous solution in the cuvette. All data were taken at 200C, unless otherwise stated.

The fluorescence of the near-infrared dye was quenched by t~L additions of quencher. Reproducible addition of quencher was achieved by using an EDP2 battery-op- erated, motorized micropipette (Rainin Instrument Co., Woburn, MA). Care was taken to add the quencher while the stirrer was on, to minimize concentration differences. Absorbance measurements were taken before and after the addition of quencher to ensure that the dye did not undergo chemical reactions. Absorbance values used in these experiments were obtained on a Varian DMS 200 UV-visible spectrophotometer.

RESULTS AND DISCUSSION

Table II qual i ta t ively summarizes the observed quenching of different laser dyes. Deionized water was used as solvent except when the aqueous solubility of the dye was too low. In these cases ethanol was used, as indicated in Table II. As can be seen, several of the near- infrared laser dyes were found not to be particularly sensitive to quenching processes. The results show that KI, nitromethane, and Cu(II) ions generally quenched most of the near-infrared florophores. The quenching constant is a function of the diffusion coefficient of the quencher to the fluorophore, as well as the fluorescence lifetime. Unfortunately, the lifetimes of near-infrared laser dyes are relatively short, ~4 and the lifetime measurement of these dyes requires special equipment. This relatively short lifetime explains the relative insensitivity of several near-infrared laser dyes to collisional deacti-

TABLE III. Quenching data for dyes exhibiting linear quenching behavior.

Dye/quencher Ksv [M -1] IR-140/Cu(II) 6740 IR-144/Cu(II) 20 HDITCP/Cu(II) 2050 DTTCI/CH3NO2 4.4 DTDCI/trimethylamine 48

vation. Although the short lifetimes of the near-infrared emitters limit the power of quenching studies, useful information can be obtained with respect to how the different dye structures will determine the stability of near-infrared labels.

We have observed linear and nonlinear Stern-Volmer quenching processes. Several of the near-infrared laser dyes exhibited linear or quadratic behavior. The least- squares values which describe the linear curves are presented in Table III. The pronounced quenching observed demonstrates that the traditional Stern-Volmer equation can be used to monitor the quenching phenomenon and the sensitivity of quenching of these particular near- infrared dyes. No significant changes in the fluorescence emission profiles have been observed. Changes in the emission profile would indicate quencher-induced perturbations in the fluorescence electronic transitions. This qualitative information is valuable in detecting the effect of quencher on the near-infrared fluorophores.

The quadratic behavior may be described under the assumption of combined dynamic and static quenching processes. 15 For combined static and dynamic quenching interaction, a concave curvature is usually observed in the Stern-Volmer plot. However, it should be noted that this concave curvature is not absolute proof for the presence of combined static and dynamic quenching, since heterogeneous emission systems can also result in a concave plot. TM Nevertheless, the presence of multiple emit- ting centers in our case is not very likely. No spectral changes were observed that characterize the aggregation of near infrared dyes. iv Partial solubilization was also discounted because the dye concentration was below the solubility limit. Hence, we can assume the presence of combined static and dynamic quenching.

DTTCI. Nitromethane and Cu(II) were quenching this dye. According to our data (Table IV), Cu(II) is a static quencher for this dye, since the Stern-Volmer constant is bigger at lower temperature. This is expected since Cu(II) may form a ground complex with this dye. This assumption is confirmed by the absorption spectra. The fluorescence of the DTTCI was quenched by nitromethane at room temperature. At 0.5°C, practically no

TABLE IV. Quenching data for DTTCI.

Quencher Temperature [°C] Ksv [M ~] Cu(II) 0.5 1.45 × l0 s

35 1.04 x l0 s Nitromethane 0.5 ~0

20 4.4

1452 Volume 43, Number 8, 1989

1.41

1.34

1.27

1,2C

1.14

1.07 "

1.00 '

H I T C P - KI

I

0.(30 5.05 10.11 15.16 20,21 25.26 30.32 35.37 Concentrat ion (raM)

e.36 -~

7.32

6.27

6.21

4.16

3.11

2.05

1.00 q O.O0

A

t R - 1 2 5 - C u ( l l )

0.21 0.42 0 .62 0 .03 1.04 1.20 1.40 Concentration (raM)

7 ,55

6.44

6.53

4.63

H I T C P - C u ( l l )

3 .72

2.81

1.91

1,00 , l , *

O.O0 0.46 0.92 1.36 1.84 2.30 2.77 3.23 Concentration (raM)

FiG. 1. Stern-Volmer plots of fluorescence quenching for HITCP with KI and Cu(II).

quenching was observed, indicating possible dynamic quenching process.

HITCP. The fluorescence of HITCP was quenched by KI and Cu(II) ions. Both quenchers resulted in nonlinear Stern-Volmer plots (Fig. 1). However, when ( F o / F - 1)/ [Q] was plotted as a function of the quencher concentration, more linear plots were obtained. This may indicate a combined dynamic and static quenching process. However, no Stern-Volmer constants (dynamic and static) could be extracted from these plots because it would result in negative value for one of the constants. This observation indicates a more complex process. KI has been shown to be a collisional quencher. It is quite sur- prising that we have observed nonlinear quenching curves. A secondary quenching process in this study can be explained if the counterion of the laser dye (perchlorate) is replaced by the excess I- ions. The positive charge of the laser dye area adjacent to the chromophore concen- trates the quencher I- ions. The excited fluorophore is then collisionally deactivated before it can fluoresce.

IR-144. The results show that only the Cu(II) ion quenches this laser dye. However, the observed quenching was very small (Ksv = 20 M -1) at room temperature. No significant change was observed at lower or higher temperatures (0.5°C and 35°C, respectively), making the distinction between static or dynamic processes impos- sible. The relatively low degree of quenching in the present study may be explained if the chromophore of IR-

5060

4385

3680

2975

I

2271

861 •

/ 166

0.17

I R - 1 2 5 - C u ( l l )

, * , ~ , ,

o .35 0.54 o.72 o.91 1 ,o9 1,26 %46 Concentrat ion (raM)

FIG. 2. Stern-Volmer plots of fluorescence quenching for IR-125 with Cu(]I).

144 is protected by the relatively bulky substituents around the chromophore.

IR-140. This dye exhibited very similar behavior to that of IR-144. The results indicate that only Cu(II) quenches IR-140. Nevertheless, the observed quenching was much higher. The fluorescence quenching shows Stern-Volmer constants of 3820 at 2~C and 6740 at room temperature, respectively. At higher temperatures (35°C), the quenching became so significant that it is difficult to determine the exact Stern-Volmer constant. It is inter- esting to compare the molecular structures of IR-140 and IR-144. Although both molecules are fairly bulky, several major differences can be observed. The much higher degree of quenching may be explained if the chromophore is more accessible to the quenching species. The chromophore portion of the IR-140 molecule is much less protected than that of the IR-144.

IR-125. The use of this dye has recently received some attention in the area of separation scienceY '3 Protein in human serum was labeled and the complex was separated by a gel filtration column. 2-Nitro-l-propanol, KI, and Cu(II) quenched the fluorescene for IR-125. Results indicate that it is unlikely that the zwitterionic structure of this near-infrared dye itself influences the quenching results. (Note that IR-144 also has a zwitterionic structure.) The similarities and the differences for the Stern- Volmer values of the different near-infrared laser dyes indicate that the exposure of the chromophore is a more


I R - 1 2 5 - 2 N ~ I P r O H

6.85 l

5.87

4.90 [

2.05 l

1.97 l

1.00 O.O 26.0 52.0 77.9 103.9 129.9 155.9 181.9

Concentro6on (raM)

I R - 1 2 5 - KI 1.34

1.29

1.25

1.20

1.15

1.1o

1.05

1.00 ~ t i i t i O.oo 0.46 0.92 138 1.84 2.30 2.76 9.29

Concentrotion (roB)

I R - 1 2 5 - 2 N - 1 P r O H 37.52

32.40

o 27.29

22.17

~ 17.05

11,94

. i 18.7 42.0 65.3 ~8.6 112.0 1353 158.6 181.9

B Concentrotion (raM)

FIG. 3. Stern-Volmer and moditied Stern-Volmer plots of fluorescence quenching for IR-125 with 2-nitro-l-propanol.

important factor. Although nonlinear quenching curves were obtained, Cu(II) was able to decrease the fluorescence from IR-125 (Fig. 2A). The quenching results with Cu(II) should, however, ]be interpreted in light of the nonlinear quenching curw~ obtained. The ( F o / F - 1)/[Q] plots resulted in a more linear relationship, indicating possible combined dynamic and static quenching. The individual quenching constants cannot be determined from these plots, since pronounced upward curvature is observed (Fig. 2B). Similarly, the 2-nitro-l-propanol quenched the fluorescence from the IR-125 in a nonlinear manner (Fig. 3A). Again, the ( F o / F - 1)/[Q] plots did not reveal further information, due to their extreme non- linearity (Fig. 3B). These nonlinear plots indicate that the quencher interaction with the fluorophore may not be described as being simple dynamic or static quenching or the combination of both. Undoubtedly, the quencher produces other interactions which affect the fluorescence intensity of this near-infrared laser dye. We should not rule out completely that changes in the hydrophobicity of the aqueous media upon addition of this quencher may decrease the dimer concentration of IR-125, although no dimer band was observed. Near-infrared dyes have been shown to dimerize in aqueous solutions over a certain concentration range. 17 The fluorescence of IR- 125 was quenched by KI. The Stern-Volmer plots also indicate complex processes in the case of this quencher (Fig. 4). The secondary quenching process is not as sig-

1454 Volume 43, Number 8, 1989

106.3

93.3

60.3

67.2

54.2

41.1

28.1

15.1 ~ i i , =

0.33 0,75 1.16 1.57 1.09 Concentro6on (raM)

I R - 1 2 5 - El

2.40 2.81 3.23

FIG. 4. Stern-Volmer and modified Stern-Volmer plots of fluorescence quenching for IR-125 with K1.

nificant as it is in the case of HITCP. This is expected since the IR-125 molecule is not able to concentrate I- ions.

HDITCP. This dye structure is very similar to that of the IR-125, except that there is no zwitterionic structure. This difference in molecular structure results in an over- all lower susceptibility to the quenchers tested (Table II). Accordingly, only Cu(II) quenched the fluorescence of the HDITCP in these experiments. The Stern-Volmer plots which describe the fluorescence quenching for the HDITCP are shown in Fig. 5. Although nonlinear quenching was observed, the ( F o / F - 1)/[Q] plot revealed that the secondary quenching process is present only at higher quencher concentrations (Fig. 5B). Hence, we re- port the least-square fit in Table III.

DTDCI. Addition of trimethylamine did decrease the fluorescence intensity of this near-infrared laser dye. The Stern-Volmer plots for fluorescence quenching of DTDCI give fairly linear relationship. The Stern-Volmer constant was 48 for trimethylamine.

CONCLUSIONS

Fluorescence quenching measurements provided useful data for studying near-infrared fluorophores. Fluo- rescence quenching supplies additional information on the fluorophore by specifically introducing a chemical probe. Since near-infrared fluorophores have found an

HDITCP - Cu(ll) 4.63 T

4,11 [

3.59

3.07 l

2.58 1

2.04 f

1.52 ~

1.C~3 i i i i , ~ 0.00 0,25 0.46 0.69 0.92 1.15 t.38 1.61

Concentrotion (raM)

3130

2941

2752

o "~ 2563 i i

~ 2375

2184

1995

1806

HDITCP - Cu(ll) i

i

D.17 0.~7 0.58 0.78 0.99 Concentration (raM)

1,20 1.40 1.61

FIG. 5. Stern-Volmer and modified Stern-Volmer plots of fluorescence quenching for HDITCP with Cu(II).

increasing application as labeling agents in different areas of chemical analysis, it is important to study the influence of different quenchers. The effect that certain quenchers have on these very short-lived fluorophores is substantial. KI produces a large quenching on the fluorescence of several near-infrared dyes, while Cu(II) ions have the greatest effect. The quenching effect of Cu(II) seems to be connected to the capacity for ground-state complex formation of near-infrared laser dyes. Cu(II) ions produce significant changes in the fluorescence intensity of near-infrared fluorophores. These changes are

produced by a combination of effects on molar absorptivity, quantum yield, and possible lifetime of the near- infrared fluorophore. The mechanisms of these interactions have not been fully deduced, and further study of these systems is ongoing. One of these ongoing studies is the evaluation of microenvironmental effects on com- plexed near-infrared fluorophores (e.g., protein complexes).

These results may prove useful in future analytical procedures where near-infrared dyes could be used to selectively detect near-infrared dye derivatized molecules. Near-infrared fluorophores may also prove to be useful in other bioanalytical determinations, where the increased sensitivity and the long wavelength of detection would improve detection limits and decrease the need for preseparation methods. Studies using near-infrared dyes as labeling agents are currently under de- velopment.

ACKNOWLEDGMENT

Acknowledgment is made to the Donors of Petroleum Research Fund, administered by the American Chemical Society, for support of this research.

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(1986). 4. E. G. Arthurs, D. J. Bradley, and A. G. Roddie, Appl. Phys. Lett.

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Chem. Phys. Lett. 18, 488 (1973). 7. V. A. Kuzmin and A. P. Darmanyan, Chem. Phys. Lett. 54, 159

(1978). 8. W. Cooper and K. A. Rome, J. Phys. Chem. 78, 16 (1974). 9. A. A. Muenter and W. Cooper, Chem. Phys. Lett. 22, 212 (1973).

10. J. P. Fouassier, D. J. Lougnot, and J. Faure, Opt. Comm. 18, 263 (1976).

11. N. Fletcher, Appl. Phys. 22, 227 (1980). 12. K. H. Feller, D. Faessler, P. Hampe, K. Berndt, E. Klose, and P.

Schwarz, Z. Phy. Chem. 266, 2 (1985). 13. D. E. Wolf, Biochemistry 24, 582 (1985). 14. M.I. Demchuk, A. A. Ishchenko, V. P. Mikhailov, and V. I. Avdeeva,

Chem. Phys. Lett. I44, 99 (1988). 15. J. R. Lakowitz, Principles of Fluorescence Spectroscopy (Plenum

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Fluorescence Quenching Studies of Near-Infrared Fluorophores