FLUORESCENCE | Clinical and Drug Applications☆

A Gómez-Hens, University of Cordoba, Cordoba, Spain

ã 2013 Elsevier Inc. All rights reserved.

Introduction 1
Selectivity and Sensitivity of Fluorescence Spectroscopy in Clinical and Drug Analysis 1
Chromatographic and Electrophoretic Methods 2
Enzymatic Assays 5
Fluoroimmunoassays 6
Fluorosensors 7
Proteomics Studies 7
Fluorescent Nanoparticles 8
Other Applications 8

Introduction

The usefulness of fluorescence spectroscopy for clinical and drug analysis is beyond doubt. In fact, judging by the number of
analyses done per day, the most extensive use of this technique is probably in clinical and pharmaceutical analysis. A large number
of substances of biomedical interest possess intrinsic fluorescence at either room or low temperature, which has been widely
exploited for performing basic studies and developing a host of analytical methods. Also, many therapeutic and abuse drugs exhibit
native fluorescence in organic and neutral solutions or even in strong acids and bases, which has been used for analytical purposes.
Nonfluorescent or weakly fluorescent analytes can be modified to obtain fluorescent products. The changes involved in the
process can be of chemical or physicochemical nature. The former, usually in the form of derivatization reactions, include chemical
treatments using redox, complex formation and substitution reactions or incorporation of a fluorophor by chemical (e.g.,
condensation) reaction. Physicochemical modification of a nonfluorescent or weakly fluorescent substance can be accomplished
in a number of ways including changes in the molecular environment arising from a solvent changeover, addition of a chemical
compound such as a surfactant or cyclodextrin to protect fluorophores from their molecular environment, and energy transfer
processes between fluorescent and nonfluorescent molecules.
A systematic description of the scope of fluorescence spectroscopy in clinical and drug analysis is rendered difficult by the very
large number of determinations reported so far. For reasons of space, this article can only present an overview of the wide variety of
applications in these analytical fields, such as chromatographic and electrophoretic methods, its use in enzymatic and immuno-
chemical assays and in biosensors, and its usefulness in new research areas such as proteomic studies. Also, the relatively recent use
of fluorescent nanomaterials, mainly quantum dots, in several clinical and drug applications will be briefly described.

Selectivity and Sensitivity of Fluorescence Spectroscopy in Clinical and Drug Analysis

Direct fluorescence measurements in biological samples are hindered by several factors including concentration effects, back-
ground effects arising from Rayleigh or Raman scattering, solvent effects (e.g., interfering nonspecific fluorescence and quenching)
and sample matrix effects. Thus, a serum or urine sample may contain a variety of fluorescing substances, such as proteins and
bilirubin, that are a potential source of unwanted background fluorescence. Light scattering by proteins and lipoproteins can also
contribute to this negative effect. Selectivity is also a major problem in the determination of drugs in their dosage forms, which are
often multicomponent preparations. The lack of selectivity in fluorescence analysis of clinical and pharmaceutical samples usually
calls for a prior sample clean-up step using a separation technique, such as liquid-liquid or solid-phase extraction. Dialysis is used is
some cases to deproteinize serum. As regards sensitivity, the facts that many drugs are administered in very low doses and that
biological fluids usually contain a large number of substances at low concentrations call for highly sensitive procedures.
The selectivity and sensitivity of fluorimetric analysis can be enhanced by use of a number of alternatives to conventional
fluorimetry. The usefulness of long wavelength fluorophores in avoiding the interference of the sample matrix has been shown in
numerous applications. Advances in fluorescence instrumentation allow interferences to be minimized or even eliminated
altogether, thereby providing higher resolutions. Stray and scattered light can now readily be avoided. Also, sample preparation
and handling techniques have been improved in parallel, and a number of enhanced spectral calibration and correction procedures
have been developed. In addition, computerized fluorimetric instrumentation endows assays with higher precision and obviates
the need for interpretation of vast amounts of data.

☆
Change History: March 2013. A Gómez-Hens has added keywords and abstract. References updated, and various updates to paragraphs in the article.

Reference Module in Chemistry, Molecular Sciences and Chemical Engineering http://dx.doi.org/10.1016/B978-0-12-409547-2.00152-9 1

2 FLUORESCENCE | Clinical and Drug Applications

Chromatography

High-resolution Capillary
methods Clinical
electrophoresis
applications of
laser-induced
fluorescence
spectroscopy
Enzymatic and
Flow cytometry immunochemical
methods
Fiber-optic-based
sensors

Figure 1 Scope of application of LIF spectroscopy in clinical analysis.

The determination of extremely low concentrations call for laser-induced fluorescence (LIF) spectroscopy, which is hardly
affected by stray light thanks to the high monochromaticity of the beams used. Although laser instruments are still far from
commonplace in the clinical laboratory, they have been used in a number of interesting applications (Figure 1) such as
chromatographic and electrophoretic methods, since the good collimation of the laser beam allows its full intensity to be focused
onto a very small area. Also, the detection limits of laser-based enzymatic and immunochemical methods are typically lower than
those afforded by conventional radiation sources. Other interesting applications of LIF in biomedical analysis involve the use of
fibre-optic sensors, which enable in vivo measurements, and the application of fluorescence line-narrowing spectrometry, a low-
temperature solid-state spectroscopy, to a variety of biomolecular systems including cellular macromolecular damage and chemical
carcinogenesis. Lasers are also very useful in fluorescence microscopy and flow cytometry.
Fluorescence spectroscopy has been used in a number of classical diagnostic tests, the capabilities of many of which have been
enhanced by exploiting the advantages offered by the development of fluorimetric instrumentation. Thus, screening of newborns
with normal tyrosine levels for phenylketonuria and hyperphenylalaninaemia is routinely carried out using fluorimetric assays.
Fluorimetric procedures for noradrenaline, adrenaline, dopa, dopamine and their metabolites allow the specific type of catechol-
amine and its concentration to be determined, which in turn permits characterization of pheochromocytomas and neuroblasto-
mas. Many vitamins and their metabolites are best quantified for diagnosis of avitaminosis by fluorimetric assay. The mineral
metabolism of calcium and magnesium is studied fluorimetrically in many laboratories. Fluorimetric assays for porphyrins are very
simple and sensitive. Adrenocortical function is usually assessed by using a fluorimetric procedure for corticosteroids. Also, urinary
estrogens are typically assayed fluorimetrically in clinical laboratories.
Although fluorescence spectroscopy is usually applied in solution, fluorescence measurements on solid surfaces are becoming a
major choice for screening of drugs of abuse, pharmaceuticals and clinical substances. In addition to thin-layer chromatographic
plates, other solid-surface attachments such as electrophoresis steps, silicone rubber pads and sodium acetate layers can be used for
this purpose. Also, a number of fluorimetric methods use immobilized enzymes or immunoreagents. In this particular analytical
field, measurements can be made on specially designed fluorimetric pads containing all the reagents required for quantitative assay,
with substantial time savings.

Chromatographic and Electrophoretic Methods

Fluorescence spectroscopy has reached a prominent position among the detection systems used in conjunction with separation
techniques, mainly liquid chromatography (LC) and capillary electrophoresis (CE). The literature abounds with references to
clinical and pharmaceutical applications of LC with fluorimetric detection. Thus, as can be seen in Table 1, a host of methods for
the determination of therapeutic and abuse drugs using LC and native fluorescence measurements have been described. There are
also numerous methods for drug determination in clinical samples involving pre- or post-column derivatization reactions
(Table 2). Fluorescence labeling through covalent binding is the type of reaction most commonly used in this context. Post-
column photochemical reactions induced by ultraviolet irradiation are another useful means for converting some substances such
as phenothiazines into strongly fluorescent products. Another possibility is to exploit the dependence of the emitted radiation
intensity on the particular environment of a fluorescent compound, which is added to the eluent. Thus, lipids have been
determined by mixing a continuous stream of aqueous solution containing a fluorescent dye with the column effluent, which
results in a certain level of baseline fluorescence that is increased by the presence of lipids, probably because of the enhancing effect
of the micelles formed by the lipids.
LC with fluorimetric detection is also suitable for therapeutic drug monitoring (TDM) purposes. For instance, it has been
applied to the determination of selective serotonin reuptake inhibitors, which are used as antidepressants. Table 3 shows some
 FLUORESCENCE | Clinical and Drug Applications 3

Table 1 Drug families determined in clinical samples using LC and intrinsic fluorescence.

Family Compound Sample Family Compound Sample

Aminoacids Tryptophan Brain tissue, Antituberculous agents Aminosalicylic Acid Plasma,

Tyrosine plasma, urine Blocking agents Acebutorol urine
Plasma (b-adrenoceptors) Plasma,
Analgesics Ibuprofen Serum Alprenolol serum, urine
Naproxen Plasma Atenolol Plasma
Salicylamide Bunobronol Plasma
Plasma, serum, urine and others Plasma, urine
Antibacterial agents Ciprofloxacin Plasma, Celipronol Plasma, urine
urine Plasma, urine
Plasma, Metopronol Plasma, urine
Norfloxacin urine Plasma, urine
Serum Penbutorol Plasma, urine
Ofloxacin Plasma, urine Plasma, urine
Plasma Pindolol Plasma, urine,
Pefloxacin Plasma saliva
Difloxacin Propranolol Plasma
Plasma Plasma, urine
Sulphapyridine Plasma Sotalol Plasma, urine
Sulphasalazine Plasma Plasma, urine
Anticonvulsants Plasma Dermatological Salicylic acid Urine
Antidepressants Carbamazepine agents
Desipramine Serum Plasma
Imipramine Diuretics Amiloride Plasma,
Trimipramine Serum Bumetanide urine
Antidiabetics Plasma,
Glyburide Plasma Furosemide urine
Antihelmintics Plasma Serum,
Thiabenzasole Plasma Metolazone Urine
Antihypertensives Blood
Indoramin Plasma, Hallucinogens Lysergic acid Blood
Labetalol urine diethylamide Plasma
Prazosin Plasma Narcotic analgesics Codein
Antimalarials Sympathomimetics Prenalterol
Chloroquine Plasma,
urine Tranquillizers Pipothiazine
Pyrimetamine Plasma
Antineoplastics Plasma Sulpiride
Daunorubicin
Vitamins Pyridoxine
Dexorubicin Riboflavin
Melphalan Tocopherols

features of these methods, which involve a sample pretreatment by liquid-liquid or solid-phase extraction. An interesting appli-
cation of LC with fluorimetric detection to drug analysis is the separation of enantiomers. For instance, cardiovascular agents
represent one of the largest prescribed class of pharmaceuticals since cardiovascular diseases are one of the main causes of mortality
in the world. Many of these compounds are still administrated as a racemic mixture, although the (R)- and (S)-individual
enantiomers usually possess different pharmacological and toxicological activity. Table 4 shows some examples of the usefulness
of fluorimetry in this area.
Probably, the main analytical application of CE is, for the present, the determination of drug substances and, indeed, the
penetration of this technique into the pharmaceutical industry, after a slow start, is now a reality. Also, CE is becoming a tool
complementary to established LC methods for drug determination in biological samples. The separation efficacy of CE can be
greater than that of LC and CE can be less expensive because organic solvent consumption is lower and the capillaries are cheaper.
However, CE cannot yet be regarded as a routine technique in clinical and drug analysis. There are still several problems such as the
lack of sensitivity and the need for sample pretreatment to remove interfering species and particulate matter that can easily clog the
CE system. Sometimes CE is not sensitive enough to determine low concentrations because of the low sample-injection volume,
which is typically only a few nanoliters and the short optical path-length for on-capillary detection.
Fluorescence detection in CE is generally performed using laser sources for excitation instead of conventional lamps, with the
aim of concentrating enough radiation energy inside the capillary. However, although the use of LIF provides extremely high
4 FLUORESCENCE | Clinical and Drug Applications

Table 2 Examples of derivatization reactions used in LC for the fluorimetric determination of drugs in clinical samples.

Reaction/reagenta Analyte Sample

Pre/o-phthaldialdehyde Gentamicin Plasma, urine

Histamine Plasma, urine, tissues
Amphetamine Plasma, urine, tissues
Phenylpropanolamine Urine, plasma
Pre/dansyl chloride Maprotiline Plasma, urine
Perbexiline Plasma
Trimetazinide Plasma
Barbiturates Serum, blood
Cannabinoids Urine
Tocainide Plasma
Pre/dansyl hydrazine Hydrocortisone Plasma
Pre/dansylazidine Penicillamine Serum
Pre/fluorescamine Gentamicin Plasma, urine
Tocainide Plasma
Pre/7-chloro-4-nitrobenz-2-oxa-l,3-diazole Amphetamine Blood, urine
Pre/N-(1-pyrene) maleimide Captopril Plasma
Pre/naphthacylbromide Valproic acid Plasma
Pre/4-bromomethyl-7-methoxycoumarin Barbiturates Serum, blood
Pre/deacylation Indomethacin Plasma, urine
Pre/oxidation Dihydromorphine Urine
Emetine Plasma
Morphine Urine
Reserpine Plasma
Methotrexate Plasma
Post/oxidation Morphine Blood, urine
Nalorphine Blood, urine
Isoprenaline Plasma, urine
Thiamine Blood
Benzodiazepines Serum
Post/fluorescamine Cephradine Serum
Cephratricine Urine
Amphetamine Urine
Post/hydrolysis Indomethacin Plasma, urine
Post/irradiation Clobazam Serum
Demoxepam Serum, urine
Penbendazole Serum
Phenothiazines Serum, urine
Clomiphene Plasma
Stilboestrol Urine
Cannabinoids Urine
a
Precolumn (Pre) or postcolumn (Post) derivatization

Table 3 Features of methods for therapeutic drug monitoring of selective serotonin reuptake inhibitors.

Analytes Sample Extractiona Derivatization LOQb(ngml
1)

Citalopram Plasma LL No 5
Desmethylcitalopram
Didesmethylcitalopram
Citalopram propionic acid
Fluoxetine Serum SPE No 20
Norfluoxetine Plasma LL Yes 3
Fluvoxamine Plasma LL Yes 3
Paroxetine Serum SPE No 5
a
Liquid-liquid (LL) or solid-phase (SPE) extraction.
b
Limit of quantitation.
 FLUORESCENCE | Clinical and Drug Applications 5

sensitivity, it is only applicable for some analytes as lasers are commercially available only with a limited number of wavelengths.
For instance, some drugs like anthracyclines fluoresce when excited at 488 nm, which is the typical emission wavelength of the
argon ion laser. Thus, detection limits of 0.5 and 2 ng ml
1 have been obtained for idarubicin and doxorubicin, respectively, using
a sample volume of 0.1 nl plasma with a commercial CE instrument equipped with an argon laser. The use of a He–Cd laser
emitting at 325 nm has allowed also the determination of several drugs, such as ciprofloxacin, methotrexate and zolpiden, in
biological fluids by CE-LIF. An alternative to direct fluorescence detection is the use of a derivatization reaction although it can
involve a loss of selectivity. In spite of these limitations, the use of LIF detection is one of the preferred options for improving
sensitivity in the analysis of biological samples using CE. The interest in the near-infrared region in CE using diode lasers and long-
wavelength fluorophores has grown in the last years as it is a useful option for avoiding background interferences. This approach
has been applied to the determination of amino acids, peptides and proteins, and DNA sequencing and fragment analysis.
The usefulness of LIF in DNA analysis has been demonstrated using fluorescent dyes as labels in CE and denaturing LC
(DHPLC) techniques. It has been suggested that CE is preferable for DNA sizing and sequencing analyses and DHPLC is effective
for screening of genetic mutations and polymorphisms.

Enzymatic Assays

The high sensitivity of fluorescence spectroscopy and the selectivity of enzymatic assays are responsible for the increasing use of
fluorimetric methods in enzymology. Enzyme determinations usually involve the use of kinetic methodology for measuring the
rate of formation of the fluorescent product, while both equilibrium and kinetic methods are used to determine the substrates.
Fluorimetric measurements on enzyme-catalysed reactions have been used for a long time to determine a variety of enzymes and
substrates (Figure 2).
NADH and NADPH, the respective reduced forms of NADþ and NADPþ, are highly fluorescent; hence, all NADþ- and NADPþ-
dependent reactions involved in enzymatic assays can be monitored fluorimetrically with a sensitivity higher than those of

Table 4 Chromatographic enantioseparation of cardiovascular drugs using

fluorescence detection.

Analyte Sample

Sotalol Plasma, urine

Propranolol Urine
4-Hydroxypropranolol Urine
Albuterol Serum
Verapamil and norverapamil Plasma
Gallopamil Plasma
Mexiletine Serum
Ibutilide Plasma
Artilide Plasma

Deaminases Acids
Aminoacids
Decarboxylases Bile acids
Enzymes Organic acids
Dehydrogenases
Acetylcholine and choline
Hydrolases
Alcohols
Kinases
Substrates Amines
Lyases
Carbohydrates
Oxidases
Cholesterol
Transaminases
Creatinine and creatine

Glycerol and triglycine

Urea

Xanthine, hypoxanthine
and uric acid

Figure 2 Fluorimetric determinations based on enzyme-catalyzed reactions.

6 FLUORESCENCE | Clinical and Drug Applications

absorptiometric techniques by two or three orders of magnitude. Reactants must be used at much lower concentrations, and
detection limits of 10-9 mol l
1 or even lower can be achieved. Numerous methods for enzyme determination involve the use of
substrates that are transformed into highly fluorescent products. For instance, p-hydroxyphenylacetic acid has been used widely as a
substrate for the determination of several oxidases such as glucose oxidase and xanthine oxidase. Enzymatic substrates yielding
fluorescent products that emit at long wavelengths are a useful alternative for avoiding background signals. Thus, naphthofluor-
escein diphosphate has been proposed as substrate for alkaline phosphatase, which hydrolyses the substrate yielding fluorescent
naphthofluorescein. This system has been used for the determination of theophylline at therapeutic levels based on the inhibitory
effect of this drug on the enzyme activity. Fluorogenic substrates for proteases containing Cresyl Violet have been synthesized to
visualize protease activity in single cells. Also, a tetra-substituted amino aluminium phthalocyanine has been described as a red-
region substrate for peroxidase. The system has been applied to the determination of glucose in serum by coupling a glucose
oxidase-catalized reaction.

Fluoroimmunoassays

Immunochemical methods are routinely used in the clinical laboratory on account of their special features, particularly their high
selectivity and the commercial availability of automatic instrumentation for their implementation. Since the earliest immunoassay
involving radioisotopes was reported, a large variety of alternative immunoassays have been developed to circumvent the
shortcomings of radioimmunoassay, many of them using fluorescence detection. Some fluoroimmunoassays (FIAs) developed
with this purpose, such as fluorescence-quenching and fluorescence-enhancing immunoassays, are scarcely sensitive enough or
applicable, which hinder their use by clinical laboratories. However, there are some FIAs widely used in clinical analysis at present
by using automated instrumentation.
Fluorescence polarization spectroscopy is applied in homogeneous immunoassay for the determination of small molecules
such as hormones and therapeutic and abuse drugs (Table 5). It is routinely used in TDM to quantify drug levels in plasma and
serum specimens. The most serious problem faced in applying fluorescence polarization immunoassay (FPIA) to serum samples is
its relatively poor sensitivity, which is limited by three factors: (1) the background fluorescence of serum samples, which arises
partly from scattered light and partly from the intrinsic fluorescence due to the presence of fluorescent compounds such as NADH
and bilirubin; (2) nonspecific binding of the tracer to serum proteins such as albumin, which is probably the chief agent
responsible for this interaction because of its versatile ligand properties and affinity for many anionic dyes (e.g., fluorescein, the
most commonly used label in FPIA); and (3) the presence of polarizers in the excitation and emission beams, which reduces the
light intensity and potential sensitivity by a factor of up to 10. In spite of these shortcomings, the advent of microprocessor
technology, improvements in optics and detectors and advances in tracer design and immunological techniques have turned FPIA
into a practical technique for use in the clinical laboratory.
Fluorescence energy transfer immunoassay is another homogeneous format based on a nonradiative fluorescence resonance
energy process. One of the immunoreagents is labeled with a donor fluorophore, while another immunoreagent is labeled with an
acceptor chromophore, which may or may not be fluorescent. There should be considerable overlap between the emission
spectrum of the donor and the absorption spectrum of the acceptor. The formation of the antigen-antibody complex gives rise
to the quenching of fluorescence of the donor, while the fluorescence of the acceptor increases if the acceptor is fluorescent. Besides
fluorescence intensity changes, the energy transfer process also induces fluorescence lifetime changes, which can be measured by
using pulsed or modulated excitation sources. Because the fluorescence properties of the unbound species essentially remain
unchanged, homogeneous formats are possible.
Among homogeneous fluoroimmunoassays based on conventional fluorimetry, substrate-labeled fluoroimmunoassay is
another alternative for both hapten and protein determination using a fluorescent label. As in other homogeneous competitive
immunoassays, the sensitivity is limited by the serum background fluorescence and the limited amount of tracer that can be used.
The combined use of time-resolved fluorescence spectroscopy and lanthanide (particularly europium(III)), chelates as labels
has given rise to time-resolved fluoroimmunoassay (TRFIA). It provides a high sensitivity as a result of several assets of europium
chelates, such as their large Stokes shifts, which make separation of the excitation and emission wavelengths very easy; their narrow
emission bandwidths, which allow the use of narrow bandpass emission filters with no loss of energy; and the long life of the

Table 5 Scope of application of FPIA in clinical analysis.

Therapeutic drugs Illicit drugs Hormones

Antibiotics Amphetamines Thyroid agents

Antiarrhythmics Barbiturates Steroids
Anticonvulsants Cannabinoids
Antiasthmatics Benzodiazepines
Cardiac glycosides Opiates
Antineoplastics
Tricyclic antidepressants
 FLUORESCENCE | Clinical and Drug Applications 7

fluorescence they emit, such that the short-lived (l-20 ns) background fluorescence of serum, solvents, cuvettes and reagents
obtained upon flash excitation of the sample can be allowed to decay so that the only possible fluorescent signal will be due to the
europium chelate (500 ms). As a result, interfering background fluorescence, the greatest hurdle for other FIAs, is reduced by two
or three orders of magnitude. TRFIA requires the use of a xenon flash lamp or a laser excitation source to obtain time-resolved
measurements. The process is carried out in two steps: the reagent (antigen or antibody) labeled with a nonfluorescent europium
chelate is subjected to the immunoreaction, and then the europium is released and bound to another chelating agent, which
produces an intense fluorescence arising from an energy-transfer process. This requires the use of coated tube technology, in
which usually a europium-labeled antibody reacts with the immobilized antibody-antigen complex on the solid phase (i.e., the
sandwich technique). TRFIA is the basis for a host of applications, including in gynecology, thyroid disease, cancer diagnosis, viral
infections, cytotoxicity, and enzyme activity measurements. Many of these applications are now used in routine clinical analyses
implemented with commercially available kits and automated instrumentation.
Another application of lanthanide ions in immunoassay, in this case terbium(III), is enzyme-amplified lanthanide lumines-
cence. This approach involves the use of the substrate of an enzyme that does not form a fluorescent chelate with terbium(III), but
is converted to a product that does form such a chelate. Alkaline phosphatase (ALP) has been used for labeling, and several
compounds, such as salicyl phosphate, fluorosalicyl phosphate and diflunisal phosphate, as substrates. These compounds cannot
coordinate efficiently with the terbium(III)-ethylenediaminetetraacetic acid complex, but the fluorescent ternary complex is formed
in the presence of ALP, which splits the phosphate ester and releases the terbium ligand. This approach has been applied in
immunoassay to the determination of several species, such as a-fetoprotein, thyrotropin and interleukin 6, leading to very low
detection limits.

Fluorosensors

Optical sensors based on fluorescence spectroscopy and immobilized enzymes or immunoreagents (biosensors) are widely used at
present because of the high selectivity they provide. The earliest fluorosensor for glucose was developed in 1980 and was used to
measure the amount of oxygen consumed during its enzymatic oxidation by glucose oxidase in the presence of catalase, which are
covalently immobilized. Since this biosensor was developed, this methodology has been used for the determination of a large
number of species of clinical interest. Fluorosensors have also been widely applied to pharmaceutical analysis. Thus, penicillin can
be assayed by using a pH fibre sensor onto which penicillinase is immobilized. The enzyme catalyses the hydrolysis of penicillin to
penicilloic acid, and the resulting increase in the hydrogen ion concentration is monitored. Oxygen sensors based on the
immobilization of ruthenium(II) complexes, which show long wavelength fluorescence, have been modified into biosensors for
the determination of clinical parameters such as glucose or free cholesterol by immobilizing the corresponding enzyme also.
The development of antibody-based biosensors presents more difficulties than enzyme-based biosensors as the antigen–
antibody interactions are not readily reversible because of the high values of the affinity constants. Another limitation is that the
physicochemical changes resulting from the immunochemical reaction are often insufficient to provide detection limits compa-
rable with those of conventional analysis. As a consequence, indirect systems have been developed that rely on the use of enzyme-
or fluorescent-tagged reagents. Both competitive and sandwich formats are used. Evanescent wave-induced fluorescence is
frequently chosen to avoid possible interferences from the bulk media. For instance, an immunosensor based on the use of the
dye Cy5, a long wavelength fluorophor, has been described for the determination of cocaine metabolites in urine using a
competitive format.
Fiber-optic sensors involving the use of LIF measurements are of great interest in biomedical analysis as they can be used for
measuring analytes remotely, continuously, and directly within the fluid to be analyzed. This technique has been employed in
cytological applications for diagnosing tumors and atherosclerosis. The ability to focus intense beams tightly to subcellular
dimensions is significant in obtaining measurable signals from weakly fluorescent cells and essential when morphological
information is required. There is a growing interest in the miniaturization of the sensors, which allows the reduction of the sample
volume and the response time. The analytical properties of fluorescent nanosensors allow accurate analysis of biological systems at
the single cell level. To prevent cytotoxicity upon insertion of the sensor into the cell, the sensing reagent must be isolated from the
cellular environment by a biocompatible matrix that is selective to the intracellular analyte. The application of these sensors has
been greatly assisted by recent advances in digital fluorescence imaging instrumentation.

Proteomics Studies

Proteomics includes a series of key technologies that are being used to identify proteins and map their interactions in a cellular
context. With the sequencing of the human genome, the scope of proteomics has shifted from protein identification and
characterization to include protein structure, protein function and protein-protein interactions. Fluorescence-based and mass
spectrometry (MS)-based methodologies offer new capabilities in the field of proteomics through the performance of multiplex
quantitative analysis. Two-dimensional gel electrophoresis (2D-GE) is a separation technique used widely in proteomics. Visual-
ization of proteins in gels is accomplished through staining techniques. Several fluorescent stains have proved to be suitable for
incorporation into integrated proteomics platforms for global analysis of protein regulation. The linear dynamic range of detection
8 FLUORESCENCE | Clinical and Drug Applications

using fluorescent stains is usually superior to colorimetric or reverse stains. Fluorescence measurements in 2D-GE are obtained
using charge-coupled cameras or laser-based detectors as image-acquisition devices. For proteomics work, the protein stains must
be compatible with MS as the coupling of 2D-GE to MS provides both precise mass determination and protein identification.

Fluorescent Nanoparticles

The integration of nanotechnology into bioassays is a recent research area that is having a great impact. Nanoparticles (NPs), which
exhibit new properties that are not shown by the bulk matter, are being considered as an alternative to conventional reagents, such
as enzymes or organic molecules, often used in bioassays. Some reasons of this success are their ability to improve the features of
these assays, allowing their miniaturization and expeditiousness, reducing reagent and sample consumption, and facilitating the
performance of heterogeneous formats. NPs present a larger surface area for the display of receptors than flat surfaces and the
reactions are faster and more sensitive. They have been used as either immobilization platforms or labels for the detection of
numerous analytes such as proteins and nucleic acids, using immunoassays and hybridization assays, respectively. These new
reagents have opened new opportunities in several fields such as gene expression studies, high-throughput screening and medical
diagnostics.
Quantum dots (QDs) are inorganic semiconductor nanocrystals with interesting luminescent properties, which have been used
in numerous bioassays. They show broad excitation profiles and narrow emission peaks and can emit in a range of wavelengths by
changing their size and composition. Also, they lack of photobleaching and have long fluorescence lifetime. QDs offer better
cellular imaging results that those achieved by organic dyes in cell- or tissue-based drug studies. The usefulness of QDs has been
also shown in different individual and multiplexed immunoassays and hybridization assays. Some of these assays involve the use
of fluorescence resonance energy transfer using QDs as donors and fluorescent organic dyes as acceptor.
Other fluorescent NPs used in bioassays are dye-doped silica NPs, which consist of luminescent organic or inorganic species
dispersed inside a silica matrix. These NPs enable significant amplification of the analytical signal due to the numerous dye
molecules inside each NP. Also, the interest in the use of these NPs in bioanalysis lies in the versatility of silica in synthesis aspects
as well as surface modifications. Silica-based NPs functionalized for coupling and containing stable lanthanide chelates have been
used in several bioassays since they are photostable, present sharp emission spectra, wide Stokes shifts, long fluorescence lifetimes
and lack of inner-filter quenching. Also, the surface of these NPs can be easily modified without altering their properties
significantly. These lanthanide-doped silica NPs have been usually used for the development of fluorescence resonance energy
transfer based assays, in which near-infrared acceptors are used to avoid background signals.

Other Applications

There are other fluorimetric techniques of interest to clinical and pharmaceutical analyses. Thus, the performance of total
fluorescence spectrometry in multicomponent and complex analyses has been assessed. The overall fluorescence of human
serum and urine can be used as a diagnostic tool in clinical chemistry, particularly in view of its selectivity towards even quite
small changes in the location of the peaks and their relative intensities. This is the foundation of pattern recognition methods for
detection of pathological deviations from normal status. This technique is also suitable for rapid screening of a variety of drugs
since samples require virtually no treatment.
Derivative and synchronous fluorescence spectrometry, both individually and jointly, have been used in clinical and drug
analysis as a means of enhancing selectivity. Thus, derivative synchronous fluorescence spectrometry allows the direct determina-
tion of catecholamines and porphyrins in urine and of pyridoxal and its derivatives and cephalosporins in serum. In addition to its
use in immunoassay, fluorescence polarization spectroscopy has been employed to study macromolecules and to carry out
structural investigations of the binding of small molecules to proteins as well as to assess fetal lung maturity. It has also been
applied for single nucleotide polymorphisms genotyping and in drug discovery research using targets such as kinases, phospha-
tases, proteases, G-protein coupled receptors, and nuclear receptors. Fluorescence microscopy for characterizing biological surfaces
is increasingly being cited in the analytical literature. For instance, it is routinely used to study the location and movement of
intracellular species.
Fluorescence probes are widely used in flow-cytometric applications in the clinical laboratory. Some flow cytometers include
argon lasers and enable right-angle fluorescence, right-angle light scatter, and forward light scatter measurements. In this way, the
cellular DNA content, one of the most commonly determined parameters, can be measured fluorimetrically using DNA-specific
fluorescent dyes. Another interesting application of this approach is the determination of intracellular ionized calcium concentra-
tions. Several strongly fluorescing Ca2þ-sensitive dyes can be used for single-cell measurements. In multilaser flow cytometers,
several fluorescent dyes are used in order to correlate the intracellular Ca2þ concentration with those of cell surface antigens or
some other parameters. A number of immunofluorescence studies based on flow cytometry have been carried out by directly
conjugating a fluorophore (usually fluorescein isothiocyanate) to an antibody (direct immunofluorescence) or a second antibody
that reacts with the first (indirect immunofluorescence). This allows one to ascertain the presence of cell surface antigens.
Much work has been carried out in the development of new fluorescent probes suitable for the detection and quantification of
biologically significant molecules both in vivo and in vitro. For instance, aptamers are oligonucleotides that in a particular
 FLUORESCENCE | Clinical and Drug Applications 9

conformation exhibit molecular recognition. They show affinity for both small molecules and macromolecules. Aptamer beacons
are aptamers that incorporate a fluorescence-quenching pair, which is used to indicate a consequent change in conformation upon
binding. These compounds are selected for specific binding to a variety of molecular targets, ranging from small organics to
proteins. Their usefulness in molecular recognition has been studied using affinity chromatography, capillary electrophoresis and
biosensors.

Further Reading
1. Baeyens, W. R. G.; Keukelerie, D. D.; Korkidis, K. Luminescence Techniques in Chemical and Biochemical Analysis. Dekker: New York, 1991.
2. Haugland, R. P. Handbook of Fluorescence Probes and Research Chemicals. Molecular Probes™: The Netherlands, 1996; (also see http://www.probes.com).
3. Lakowicz, J. R. Principles of Fluorescence Spectroscopy. Springer Science: New York, 2006.
4. Goldys, E. M. Fluorescence Applications in Biotechnology and Life Sciences. Wiley-Blackwell: Hoboken (New Jersey), 2009.
5. Cruces-Blanco, C.; Garcı́a-Campaña, A. M. Capillary Electrophoresis for the Analysis of Drugs of Abuse in Biological Spicements of Forensic Interest. Trends Anal. Chem. 2012,
31, 85–95.
6. De Kort, B. J.; de Jong, G. J.; Somsen, G. W. Native Fluorescence Detection of Biomolecular and Pharmaceutical Compounds in Capillary Electrophoresis: Detectors Designs,
Performance and Applications: A Review. Anal. Chim. Acta 2013, 766, 13–33.
7. Feng, F.; Liu, L.; Yang, Q.; Wang, S. Water-Soluble Conjugated Polymers for Fluorescent-Enzime Assays. Macromol. Rapid Comm. 2010, 31, 1405–1421.
8. Virgo, P. F.; Gibbs, G. J. Flow Cytometry in Clinical Pathology. Ann. Clin. Biochem. 2012, 49, 17–28.
9. Wang, R. E.; Zhang, Y.; Cai, J.; Cai, W.; Gao, T. Aptamer-Based Fluorescent Biosensors. Curr. Med. Chem. 2011, 18, 4175–4184.
10. Rueda-Rama, M. J.; Walters, J. D.; Orte, A.; Hall, E. A. H. Fluorescent Nanoparticles for Intracellular Sensing: A Review. Anal. Chim. Acta 2012, 751, 1–23.
11. Li, Y. Q.; Li, X. Y.; Shindi, A. A. F.; Zou, Z. X.; Liu, Q.; Lin, L. R.; Li, N. Synchronous Fluorescence Spectroscopy and its Applications in Clinical Analysis and Food Safety
Evaluation. Rev. Fluoresc. 2010, 7, 95–117.