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76	Schulman et al.

where kET is the rate constant for resonance energy transfer, τD is the lifetime of the excited state of the donor molecule, R is the mean distance between the centers of the donor and acceptor dipoles, and Ro is a constant for a given donor-acceptor pair, corresponding to the mean distance between the centers of the donor and acceptor dipoles for which energy transfer from donor to acceptor and fluorescence from the donor are equally probable. Another general requirement for the occurrence of resonance energy transfer is the overlap of the fluorescence spectrum of the donor and the absorption spectrum of the acceptor. Any degree of overlap of these spectra will satisfy the quantization requirements for the energy of the thermally equilibrated donor molecule to promote the acceptor to a vibrational level of its excited singlet state. The greater the degree of overlap of the luminecsence spectrum of the donor and the absorption spectrum of the acceptor, the greater is the probability that energy transfer will take place.

The exchange (Dexter) mechanism of excitation energy transfer is important only when the electron clouds of donor and acceptor are in direct contact. In this circumstance, the highest energy electrons of the donor and acceptor may exchange places. Thus, the optical electron of an excited donor molecule may become part of the electronic structure of an acceptor molecule originally in the ground singlet state while the donor is returned to its ground singlet state by acquiring an electron from the acceptor. Exchange energy transfer is also most efficient when the fluorescence spectrum of the donor overlaps the absorption spectrum of the acceptor. Exchange energy transfer is a diffusion-controlled process (i.e., every collision between donor and acceptor leads to energy transfer) and as such its rate depends upon the viscosity of the medium. Resonance energy transfer, on the other hand, is not diffusion-controlled, does not depend upon solvent viscosity, and may be observed at lower concentrations of acceptor species. Energy transfer from the initially chemiexcited species to a suitable acceptor followed by fluorescence from the acceptor is an important process in chemiluminescence.

7. CHEMILUMINESCENCE REACTION PARAMETERS

CL reactions are commonly divided into two classes. In the type I (direct) reaction the oxidant and reductant interact with rate constant kr to directly form the excited product whose excited singlet state decays with the first (or pseudofirst)-order rate constant ks kf kd. In the type II (indirect) reaction the oxidant and reactant interact with the formation of an initially excited product (kr) followed by the formation of an excited secondary product, either by subsequent chemical reaction or by energy transfer, with rate constant kA. The secondary product then decays from the lowest excited singlet state with rate constant kg. Type II reactions are generally denoted as complex or sensitized chemiluminescence.

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If φCL is the efficiency of the chemiluminescent reaction, which is the ratio of the number of photons emitted to the number of molecules of reactant reacting in toto, it can be defined for a type I reaction as

φCL φc φE φf

(5)

where φc, the chemical yield, is the ratio of the number of molecules that react through the chemiluminescent pathway to the total number of molecules reacted; φE, the excitation yield, is the ratio of the number of molecules that form an electronically excited product to the number of molecules that react through the chemiluminescent pathway; and φf is the quantum yield of fluorescence of the light-emitting species.

In a type II reaction

φCL φc φE φET φf

(6)

where all symbols have the same meaning as above and φET is the efficiency of energy transfer from the initially chemiexcited species to the energy-transfer acceptor. φCL is, of course, a function of the chemical and photophysical factors described in Sec. 1–3 such as solvent polarity, reagent concentrations, and molecular structure.

The physical significance of φCL is that under defined experimental conditions it is the constant of proportionality between ICL, the observed intensity of chemiluminescence, and the rate of consumption of the initial luminophore (reactant L); i.e.,

ICL φCL ( dL/dt)

(7)

8. KINETIC CONSIDERATIONS

8.1 Type I Chemiluminescence Reaction

If the reactants in a type I chemiluminescence reaction are rapidly mixed they will result in an emission whose intensity ICL can be measured as a function of time. A typical time intensity curve for a CL reaction is shown in Figure 2.

The shape of the curve depends on the kinetics of the reaction. In Figure 2 the mixing process is not rate limiting and Eq. (7) is obeyed (if the mixing rate is rate limiting, the time required to achieve ICL(max) will be much longer). If the reaction is first order in analyte (L) (the oxidizing agent is in excess and kf kr) then Eq. (7) can be written as:

ICL φCL kr [L]

(8)

L is the analyte concentration as a function of time (Lo e k r t) and kr is as defined in Sec. 7. In this case the intensity, as in all kinetic methods of analysis, can be

78	Schulman et al.

Figure 2 Chemiluminescence intensity (ICL) as a function of time (t) for A, a reaction whose rate is much slower than the time for mixing of the reagents, and B, a reaction whose rate is comparable to the mixing time.

measured at a fixed time after mixing. The maximum emission intensity can be measured and related to concentration. If the total intensity-time curve is integrated as in endpoint or equilibrium analysis, the total light yield is obtained and the resulting integrated intensity will be proportional to the concentration and independent of the reaction rate. It is from the integration over the time course of the reaction that the great sensitivity of CL analysis is derived.

8.2 Type II Chemiluminescence Reaction

A type II CL reaction can be represented as sequential chemical and energy transfer reactions.

k k

L Ox →r P* P* A →ET A* →k hν A

Here, Ox is the oxidant that reacts with the reductant L, kr is the pseudo- first-order rate constant causing the rise in P*, the excited product that is the intermediate in the type II reaction, and ks is the rate constant for deactivation of A* [ks kf kd in Eq. (1)]. P* will fall in concentration with rate constant kET as it transfers electronic excitation to the acceptor A to form the excited acceptor A*.

If P is in excess two extreme situations can be distinguished:

1.If kr kET, the concentration of P* will not be depleted and ICL reflects [P*] as a function of time.

The Nature of Chemiluminescent Reactions

79

2.If kr kET, then P* is consumed as soon as it is formed and ICL reflects the rate of the oxidation-reduction reaction.

In the case the kr and kET are of the same order of magnitude, the intensity time curve can be represented as a biexponential function.

dL kr Lo (e k r t e kET t) dt kET kr

Combining Eq. (7) and (9) gives:

ICL (t) φCL kr Lo (e kr t e k ET t) kET kr

which gives the course of the type II chemiluminescent emission.

(9)

(10)

9. SUMMARY

CL is the emission of light by molecules that are excited by participation in a highly exergonic reaction, usually an oxidation. It can also be defined as the fluorescence of the electronically excited product of a chemical reaction; however, unlike fluorescence, no external light source is used.

The precursors of the light-emitting molecule must participate in a reaction that releases a large amount of energy. For luminescence in the visible spectrum (400–750 nm) 40–70 kcal/mol must be released. Only certain oxidation reactions will generate this much energy. Often, there will also be a ‘‘dark’’ reaction pathway, which may lead directly to the ground-state species, or to ground-state products that differ from those produced by fluorescence.

Subsequent to the formation of a potentially chemiluminescent molecule in its lowest excited state, a series of events carries the molecule down to its ground electronic state. Thermal deactivation of the excited molecule causes the molecule to lose vibrational energy by inelastic collisions with the solvent; this is known as thermal or vibrational relaxation. Certain molecules may return radiationlessly all the way to the ground electronic state in a process called internal conversion. Some molecules cannot return to the ground electronic state by internal conversion or vibrational relaxation. These molecules return to the ground excited state either by the direct emission of ultraviolet or visible radiation (fluorescence), or by intersystem crossing from the lowest excited singlet to the lowest triplet state.

Molecules that are involved in CL reactions are generally reduced species that can be easily oxidized, such as molecules containing amino and hydroxy groups and polycyclic aromatic ring systems. The solvent in which the experiment is carried out has a dramatic effect on the efficiency of the reaction. Solva-

80	Schulman et al.

tion can alter the shapes, the depths, and the densities of the vibrational states of the potential surfaces representing the ground states of products and reactants, as well as the lowest excited singlet state of the potential fluorophore. Structure and environment influence the rates of processes competing with fluorescence for deactivation of the lowest excited singlet state.

The quantum yield of fluorescence determines the intensity of light emission in CL reactions. The spectral wavelengths that are fluoresced depend on the structure of the molecule. Fluorescence is most often observed in highly conjugated or aromatic organic molecules with rigid molecular skeletons. Aromatic molecules with freely rotating substituents tend to fluoresce less intensely than those without these substituents. Molecular structure has a large effect on the position in the spectrum where fluorescence occurs, as well as its intensity.

Solvent interactions with solute molecules are mainly electrostatic. The differences in electrostatic stabilization energies of ground and excited states contribute to the relative intensities and spectral positions of fluorescence in different solvents. Changes in dipolar and hydrogen-bonding properties of the solute also affect the wavelength that is fluoresced. Fluorophores in solvents containing atoms of high atomic number fluoresce less intensely than those without heavy atoms, because the high atomic weight enhances the spin-orbital coupling in the lowest excited state of the solute.

Fluorescence may be decreased or completely eliminated by interactions with other chemical species in a process known as quenching of fluorescence. Two kinds of quenching may occur. The first is known as static quenching, where the interaction between the potentially fluorescent molecule and the quencher takes place in the ground state, forming a nonfluorescent complex. Dynamic quenching may also occur when the quenching species and the potentially fluorescent molecule react during the lifetime of the fluorescent molecule.

Energy transfer involves the passing of excitation energy to another molecule by a molecule in its excited state. The loss of excitation energy from the donor species results in quenching of the luminescence of the energy donor, and may result in luminescence from the energy acceptor that has become excited in the process. Energy transfer can occur by either of two processes. The resonance excitation transfer mechanism, or dipole mechanism, involves energy transfer between two molecules that are not in contact with each other, and may be separated by as much as 10 nm. The exchange mechanism, or Dexter mechanism, of excitation energy transfer is important only when the electron clouds of the donor and acceptor molecules are in direct contact. In this case, the highest-energy electrons of the donor and acceptor may exchange places. This is most efficient when the fluorescence spectrum of the donor overlaps the absorption spectrum of the acceptor.

There are two common classes of CL reactions. In the type I reactions the oxidant and reductant interact to directly form the excited product whose excited

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81

singlet state decays with the first order rate constant ks. In the type II reaction the oxidant and reactant interact with the formation of an initially excited product, followed by the formation of an excited secondary product, either by subsequent chemical reaction or energy transfer. The secondary product decays from the lowest excited singlet state with a rate constant kg. The kinetics of type I and II reactions also differ.

ACKNOWLEDGMENT

The authors are grateful to Mrs. Nancy Rosa and Mrs. Virginia Schulman for technical assistance with the preparation of this manuscript.

REFERENCES

1.KD Gundermann, F McCapra. Chemiluminescence in Organic Chemistry. Heidelberg Springer-Verlag, 1986.

2.F McCapra, KD Perring. In: JG Burr, ed. Chemiand Bioluminescence; Clinical and Biochemical Analysis. Vol 16. New York: Marcel Dekker, 1985.

3.E Lissi. J Am Chem Soc 98:3387–3388, 1976.

4.WR Seitz. Crit Rev Anal Chem 13:1–58, 1981.

5.AK Campbell. Chemiluminescence, Principles and Applications in Biology and Medicine. Heidelberg: VCH Publishers, 1988.

6.SG Schulman. Fluorescence and Phosphorescence Spectroscopy, Physiochemical Principle and Practice. London: Pergamon Press, 1977.

7.MA DeLuca, ed. Bioluminescence and Chemiluminescence, Methods in Enzymology. Vol 57. New York: Academic Press, 1978.

8.MA DeLuca, WD McElroy, eds. Bioluminescence and Chemiluminescence, part B, Methods in Enzymology. Vol 133. New York: Academic Press, 1978.

9.LJ Kricka. Clin Chem 37:1472–1481, 1991.

10.BM Krasnovitskii, BM Bolotin. Organic Luminescent Materials. Heidelberg: VCH, 1987.

11.K Nakashima, K Imai. In: SG Schulman, ed. Molecular Luminescence Spectroscopy: Methods and Applications: Part 3. New York: Wiley-Interscience, 1993, pp 1–23.

4

Recent Evolution in Instrumentation for Chemiluminescence

Dan A. Lerner

Ecole Nationale Supe´rieure de Chimie, Montpellier, France

1.	INTRODUCTION		84
2.	EVOLUTION OF NONIMAGING LUMINOMETERS		84
	2.1	Early Instruments	84
	2.2	Last Generation Instruments	85
	2.3	Bibliographic Sources for Commercial Instruments	87

3.OVERALL IMPROVEMENT IN THE DESIGN OF IMAGING

INSTRUMENTS	87
4. USER-ASSEMBLED SYSTEMS ARE STILL COMPETITIVE	88

5.CCD CAMERAS: THE HEART (AND EYES) OF IMAGING

SYSTEMS	88
6. A SIMPLE DESCRIPTION OF THE HARDWARE	89

7.KEY PROPERTIES FOR DEFINING THE PERFORMANCE

OF A CCD CAMERA		91
7.1	Introduction	91
7.2	Resolution	91
7.3	Quantum Efficiency	92
7.4	Dynamic Range	93
7.5	Dark Current	93
7.6	Noise	94
7.7	Binning	95

83

84			Lerner
8.	IMPROVING PERFORMANCE		95
	8.1	Dark Current Reduction by Cooling the CCD Chip	95
	8.2	Effect of Readout Rate on Readout Noise	95
	8.3	Back-Illuminated CCD Cameras	95
	8.4	Other Improvements	96
9.	FILL FACTOR AND THE HANDLING OF COLORS		97
10.	OTHER PROBLEMS LINKED TO HARDWARE OR OPTICAL
	DESIGN		98
11.	CHARGE INJECTION DEVICE		98
12.	COMMERCIAL INSTRUMENTS		99
13.	SOFTWARE		102
14.	CONCLUSIONS		102

1. INTRODUCTION

In recent years no truly new basic principles have been introduced for the detection of luminescence. However, the technical evolution in the field of microelectronics and optoelectronics, charge coupled device (CCD) detectors, fiberoptics, assembly techniques, and robotics resulted in the introduction on the market of new generations of instruments with increased performance, speed, and ease of handling. In this chapter, some of their typical features will be reviewed. To keep this presentation at a concrete level and to illustrate some specific item, instruments of different makes will be referred to. However, this does not imply they are better than those not cited. It is more a matter of availability of recent documentation at the time of writing. Note that numerical values cited typically relate what can be done today and may vary from one instrument to another from the same company.

Although the present chapter lays emphasis on CCD and CCD-based instruments, it begins with a section devoted to photomultiplier tubes (PMT)-based luminometers.

2. EVOLUTION OF NONIMAGING LUMINOMETERS

2.1 Early Instruments

The last three decades have seen a slow but constant evolution in the conception of instruments for chemiluminescence (CL) or bioluminescence (BL). Initially

Instrumentation for Chemiluminescence

85

users had to assemble their own luminometers but performance was not very good. Photomultipliers were used in the current detection mode, early amplifiers were noisy and affected by drift, and signals were fed to standard recorders with a slow response. Many improvements were made progressively and in different ways. At one time, researchers even tried to turn scintillation counters into luminometers, using them in a noncoincident mode to benefit from their photoncounting capability [1].

The problem of quantification of CL is better understood if the time evolution of intensity for a CL or BL reaction is analyzed. This time evolution is typically an asymmetric bell-shaped curve with a rise time that may last from much less than a second to minutes or more depending on the reaction studied. Several methods were devised to extract useful quantitative data from such a curve. The area under the curve may be integrated to define the total light emission (TL). The observation was also made that the peak intensity for CL is related to the overall rate of the light-producing reaction. So the peak height at maximum intensity may be used to quantify light emission. These basic factors, to which must be added the fact that the amount of light emitted in some important reactions is exceedingly small, explain the problems met by early experimentalists. These factors are also the ones that led the way to further improvements.

Quantification of light was first improved by the use of a printed circuit board, which allowed better reproducibility and new types of measurements. Integration of a delayed light reaction curve [2] could be carried between selected time limits (the mixing time of reagents was used to define the origin of the time scale). Besides integrators, various peak detectors were developed. Until the early nineties, many luminometers were still very simple instruments. They consisted in a PMT detector, a chamber for measurements, a sample injector (hand mixing or syringes), and the associated electronics. Direct reading was usually obtained following a simple calibrating step. Luminometers were basically analog instruments even if some used PMT in the photon-counting mode. Batch measurement was standard and typical instruments required a volume of about 1–10 mL in a test tube or a vial to carry out a determination.

2.2 Last Generation Instruments

The present generation of luminometers is composed of computeror micropro- cessor-driven digital instruments. Time limits are precisely defined by the user and controlled over a wider range (0.1–200 s or more). Even if an instrument works with the current measuring method, use of high-gain, high-impedance AC amplifiers and of the electronic nulling of the background results in high sensitivity. Cooling the PMT results in an increased dynamic range. Even if all aspects of current measuring versus photon-counting modes of operation are not yet fully understood, the photon-counting mode appears to deliver the best performance