Bioanalytical Applications of Thermal Lens Spectrometry

Mladen Franko
University of Nova Gorica, Laboratory for Environmental Research, P.O.Box 301,
SI-5001 Nova Gorica, Slovenia

INTRODUCTION

Thermal lens spectrometry (TLS) is known as one of the most sensitive spectroscopic
techniques for measurements in liquids. Owing to its inherently high sensitivity, TLS
enables measurements of optical absorbances lower than 10-6. Other important
characteristics of TLS include the possibility of probing very small (sub pL) volumes as
well as relatively fast signal response, which is on the millisecond time scale for
measurements in liquids (1) and enables TLS detection in flowing samples. The
application of TLS in routine chemical analysis is however still not widespread, due to
limited availability of suitable laser sources, particularly in the UV spectral range. This
has recently changed by the development of compact solid state lasers and high power
frequency-doubled lasers. However, the main disadvantage of TLS technique in chemical
analysis remains its limited tuneability and consequently poor selectivity. In general, TLS
instruments (2) do not offer the possibility of simultaneous multi-wavelength
measurements or spectral scanning, such as provided by diode-array spectrometric
detectors (DAD). Therefore, TLS is mainly applied as detection technique for separation
methods like high performance liquid chromatography (HPLC), capillary electrophoresis
(CE) and ion chromatography (IC), which provide the required selectivity (3). But, high
specificity can also be introduced into TLS measurements by applying the concept of
bioanalytical assays. They rely on the biorecognition capability of various biomolecules
such as enzymes and antibodies, which react or bind with the analyte with high
specificity.
Coupling of TLS to bioanalytical techniques offers important improvements in sensitivity
as well as selectivity in determination of various analytes. Still, TLS remains relatively
unknown and not frequently applied analytical technique. It is therefore the main
objective of this contribution to present TLS to a broader audience by reviewing the
applications of TLS method in bioanalytical assays, particularly those relying on
combination of TLS and flow injection analysis (FIA).
It is important to understand the concept of bioanalytical assays since the term
“bioanalytical” is frequently used for description of any analytical procedure applied for
detection of a biologically active molecule. For this reason some additional discussion
will be given in the following section on general concepts of bioanalytical assays applied
in combination with TLS.

1
On the contrary, detailed descriptions of basic TLS instrumentation will be omitted since
other texts on this subject can be found in the literature (1, 2). Similarly, the theory of
TLS for various experimental configurations has been reviewed (1, 2), and can be also
found in original papers (4-7), as well as in earlier chapters of this book.

BASIC PRINCIPLES OF BIOANALYTICAL ASSAYS

In general the bioanalytical assays used in combination with TLS can be divided into two
basic groups. The first group includes assays relying on various enzymes. In this case the
concentration of an analyte can be obtained by measuring the enzyme’s activity before
and after its interaction with a particular analyte, which inhibits the activity of the
enzyme. The analytical signal will therefore decrease with increasing analyte
concentration, as it is the case with cholinesterases (ChE) (8), which are inhibited by
organophosphorous compounds. A schematic presentation of such an interaction is
shown on Fig. 1, where the active site (the OH- group of serine) is located in a gorge,
which allows the approach only to molecules of particular configuration. The specificity
of interaction is further increased by the sequence of enzyme’s amino acids, which in the
case of acetylcholinesterase (AChE) form the so-called catalytic triad (serine, aspargine,
histidine), and with the adjacent tryptophan facilitate the approach of the specific
substrate acetylcholine. Very few inhibiting compounds can therefore react with the
active site of ChE’s, among them are carbamate and organophosphate pesticides. But,
while acetylcholine is bound to serine reversibly, organophosphates and carbamates are
bound irreversibly and therefore cause the inhibition and reduced activity of ChE’s.
Another possibility is the detection of a product formed after the interaction between the
analyte and active enzyme. In this case the analytical signal is directly proportional to the
concentration of the analyte, like for example in case of various peroxidases (9), which
can be exploited for determination of hydrogen peroxide.
The second group includes assays relying on high affinity of specific antibodies towards
their antigens (analytes), which are also known as immunoassays. In such an assay (Fig.
2) the analyte binds to primary antibodies, while labelled secondary antibodies are
subsequently attached to such an antigen-antibody complex. The analytical signal arising
directly from the labelled antibodies or from the interaction of the label (i.e. horseradish
peroxidase) with its substrate is therefore proportional to the concentration of the analyte
in the sample. A liquid phase immunoassay is however troublesome because of a difficult
separation of the free form of the antibody or antigen and their complex. Because of this
the heterogeneous immunoassays such as enzyme-linked immunoassay (ELISA) (10) are
preferred and rely on immobilization of antibodies on a solid support. Similarly,
immobilization of enzymes in other bioassays is also preferred due to a great reduction in
consumption of expensive reagents.

2
Figure 1: Schematic presentation of a specific interaction between an immobilized AChE
and an organophosphorous pesticide. Compounds of the type HX are formed as a side
product of the reaction.

A variety of solid supports can be applied for immobilization of biomolecules, including

polyurethane foam, controlled porosity glass, polystyrene beads as well as novel
materials such as monolythic CIM discs (11). Chemicofunctional nylon membranes were
recently introduced in microchips for microscopic TLS in order to incorporate some
enzymes like for example horseradish peroxidase (12). The most frequently applied
chemical procedures for immobilization are however entrapment, adsorption, adsorption
and cross-linking, covalent attachment, and copolymerization. Immobilization
procedures are complex and frequently require a sequence of additions of various
reagents and subsequent washing to remove excess reactants. Similarly, the final assays
based on enzyme activity or ELISA also require sequential additions of reagents. Such
procedures are greatly facilitated if performed in a flow mode.

3
Figure 2: Schematic presentation of interactions in ELISA shows binding of an analyte
(AN) to immobilized primary antibodies (AB1), and binding of a labelled secondary
antibody (AB2) (top). The concentration of the analyte is determined by probing the
activity of AB2 labelled with an enzyme, which transforms substrate S into detectable
product P (bottom).

FLOW INJECTION MANIFOLDS

The discussion above explains why actually all recent applications of TLS and
microscopic TLS relying on bioanalytical assays have been reported in combination with
various flow systems including flow injection analysis (FIA) (3) or microfluidic systems
on microchemical chips (13). Such combinations provide high sample throughput,
simplicity and high reproducibility as well as reduced operational costs. Furthermore, the
FIA technique provides an elegant mode of avoiding losses of sensitivity or negative
systematic errors due to degradation of photolabile analytes, exposed to high intensity
light from excitation lasers used in TLS experiments (14).
The schematic diagram of a FIA manifold used in combination with TLS is shown on
Fig. 3. Such manifolds enable several chemical reactions required for final detection of
analyte, which can be performed on-line. They might include chemical transformation of
the analyte through oxidation/reduction processes (15, 16) and/or other derivatization
procedures (colouring reactions) (14), as well as their binding to specific antibodies (17)
or enzymes (18).

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 Substrate
sample CPO
Buffer 1 COLUMN

PUMP 1 Valve Valve

1 2

Buffer 2

PUMP 2 AChE
COLUMN

Enzyme
regenerator
TLS DETECTION CELL

Figure 3: Schematic presentation of a FIA manifold with two reactors (colums) for
specific derivatisation of analytes (column 1, e.g. with immobilised chloroperoxidase -
CPO) and their recognition (column 2, e.g. with immobilised AChE).

However, the effects resulting from heat dissipation due to the mass flow out of excited
sample must be considered when TLS measurements are performed in flowing samples.
When pulsed or high modulation frequencies (some 10 Hz) with continuous wave lasers
are used for excitation, and flow rates are lower than 1 mL/min, such effects contribute to
the decrease of TLS signal in liquids by only a few percent (19, 20). But one should
consider the actual velocity of the flow and not just the flow rate, since in small detection
cells or in capillaries, such as the case in microscopic TLS (TLM), the flow velocities
can be rather high. Nonetheless, when TLS detection is performed in micro-channels
with diameters of a few ten m, and with flow rates of less than 1 L/min, which is
usually the case in TLM, the flow velocities are in the range of cm/s or lower. For such
cases the investigations of thermal lens effects in capillaries have even revealed
significant enhancements of sensitivities by moderate flows (up to 5 cm/s) (21, 22). This
effect arises from the increased thermal diffusion of the liquid in a capillary, caused by
the flow due to the edge effects. Higher thermal diffusion in turn decreases the signal rise
time, thus resulting in a higher recorded signal at a given modulation frequency.
Furthermore, the maximal enhancement is shifted towards higher flow rates as the
diameter of the capillary decreases and when the modulation frequency increases. At
higher flow velocities (over 10 cm/s for modulations at 700 Hz) the previously discussed
loss of sensitivity governs the observed TLS signals (21).

5
EFFECTS OF THERMO-OPTICAL PROPERTIES OF SOLVENTS

The fact that biomolecules used in bioanalytical assays require aqueous media for their
optimal function, brings about some important constrains related to the sensitivity of TLS
measurements. It is well known that water is not the most favourable solvent for TLS
measurements. This is due to relatively low temperature coefficient of refractive index
(dn/dT) and relatively high thermal conductivity (k) of water, compared to other solvents,
which provide much higher enhancement factors relative to conventional transmission
mode measurements of absorbance.
The enhancement factor can be written as E = P(dn/dT )/kλ, where P is the power of
excitation laser and λ the wavelength of the probe laser. Since very much different
excitation powers and probe laser wavelengths are used in TLS experiments, the
comparison of different solvents in Table 1 is based just on the ratio (dn/dT)/k. It can for
example be calculated from available thermo-optical parameters (dn/dT and k) (1) that
compared to TLS measurements in water a 39.6 times higher sensitivity can be achieved,
when TLS detection is performed in carbon tetrachloride.

Table 1: Thermooptical properties of some frequently used solvents (1) and ionic liquids.
Values for ionic liquids C8MImTf2N, BMImTf2N, EMImTf2N are calculated from data on
relative TLS sensitivity in reference 23.

Solvent k (dn/dT) 103 (-(dn/dT)/k)103

[Wm-1K-1] [K-1] [mW-1]
CCl4 0.103 -0.612 5.94
C8MImTf2N n.d. n.d. 5.25
Cyclohexane 0.123 -0.556 4.52
BMImTf2N n.d. n.d. 3.9
Acetone 0.190 -0.542 2.85
EMImTf2N n.d. n.d. 2.7
Acetonitrile 0.188 -0.45 2.39
Ethanol 0.167 -0.40 2.37
Methanol 0.202 -0.394 1.95
Water 0.598 -0.091 0.15

Unfortunately, bioanalytical assays cannot be performed in organic solvents such as CCl 4

due to denaturation of biomolecules. On the other hand, it has been demonstrated also in
some TLS applications that biomolecules such as enzymes can sustain additions of water
mixable solvents of up to 10% (24). This is important because addition of water mixable

6
solvents can be exploited to improve the sensitivity of TLS detection and to lower the
limits of detection. It has been in fact demonstrated that additions of 30% acetone,
methanol or acetonitrile can lead to up to three fold improvement of limits of detection in
case of TLS determination of chromium species by ion chromatography (25).
Application of ionic liquids as new “green solvents” was not very frequent in TLS yet. It
is however promising that enzymes such as chloroperoxidase retain high degree of their
activity even upon addition of 30% ionic liquid into aqueous solution (15, 26). On the
other hand ionic liquids were shown to cause reversible inhibition of AChE at
concentrations of just one percent of ionic liquid in aqueous solution, and can therefore
no be used for enhancement of TLS sensitivity in this case (15).
Another parameter, which can be exploited to enhance the TLS signal in aqueous
solutions is the temperature. Particularly the dn/dT of water shows noticeable dependence
on temperature (27, 28), with increasing values at higher temperatures. However,
temperatures can’t be too high since denaturation of biomolecules occurs like in case of
added organic solvents. But at moderate temperature increases, the activity of enzymes
can be enhanced as well. As already demonstrated in case of TLM, the synergistic effect
of increased enzyme activity and temperature dependent thermooptical properties of
water can lead up to ten fold increase in signal intensity when raising the temperature
from 5 to 15 C (29).

APPLICATIONS

FIA-TLS
Substantial amount of work in the field of bioanalytical applications of TLS has recently
been conducted on the utilization of the FIA-TLS for determination of organophosphate
(OP) and carbamate pesticides. For this purpose a dual beam, mode mismatched
pump/probe TLS spectrometer exploiting the Ar-ion laser as the excitation source (476
nm) was coupled to a FIA system with incorporated bioanalytical column containing
acetylcholinesterase (AChE) immobilized on glass bids (24, 30). When a substrate
(acetylthiocholine iodide) passes such a bioanalytical column it is hydrolyzed by the
action of AChE, and upon further reaction with the 5,5’-dithio-bis(2-nitrobenzoic acid)
(known as Ellman’s reaction (8)) produces a colored compound, which is detected by
TLS. The magnitude of TLS signal is therefore proportional to the activity of the enzyme
and quantification of known AChE inhibitors from the groups of organophosphates and
carbamates can be performed by determining the AChE activity before and after injection
of the sample.
The method was successfully applied for highly sensitive determination of
organophosphates such as paraoxon (LOD = 0.2 μg/L) and carbamates such as
carbofuran (LOD = 1 μg/L) in samples of tap water and spiked fruit juices, which did not
require other sample treatment than adjustment of the pH (30). Further optimization of
the method, which was dedicated to selection of the most suitable enzyme (AChE from
electric eel) (18) and development of matrix matched calibration procedures (31),
resulted in a FIA-TLS method for determination of organophosphate and carbamate

7
pesticides in samples of vegetables such as onion, and lettuce (24). Samples were
prepared by chopping, mashing in liquid N2, mixing with carrier buffer (with 10% added
acetone), centrifuging, and 1 mL of supernatant was directly injected into the FIA system
without any extraction or preconcentration step. The system was calibrated by using
paraoxon standard solutions and the concentrations of pesticides detected in each sample
were expressed in terms of paraoxon equivalents. Taking into account the differences in
the toxicities of pesticides (LD50 values for rats) present in the sample, the determined
paraoxon equivalent concentrations were reported to agree well with concentrations of
individual pesticides (carbofuran, propamocarb, oxydemeton-methyl and parathion-ethyl)
determined by GC-MS.
Other applications of FIA-AChE-TLS include monitoring of toxicity during the
photodegradation of organophosphates and related photocatalytic processes (32-35) as
well as in investigations of advanced oxidation methods for treatment of waters
contaminated by OP pesticides (36). Fast response of FIA-TLS and little sample handling
enable quasi on-line monitoring (three to five min. time resolution) of degradation
processes, which last one to two hours. Inhibition of AChE activity reveals the formation
of toxic degradation products (mainly OP oxons), which can not be detected by
monitoring the disappearance of the parent compound to determine the end point of
degradation process. Inhibitions of up to 40% of AChE activity were detected in
photodegradation and photocatalysis of malathion, isomalathion, chlorpyriphos and
azinphos-methyl with initial concentrations between 5 and 30 ppm. The inhibitions
correlated well with the concentrations of oxons (below 1 ppm) detected by GC-MS (34-
36). FIA-AChE-TLS also served as a reliable method to determine the end point (no
AChE inhibition) in degradation of oxons such as malaoxon, which were in general
found more persistent to photodegradation compared to their thio-analogs
(phosphorotionates and dithionates) (33-36). Because of much faster response and high
sensitivity for detection of oxons, the FIA-AChE-TLS was clearly found advantageous
(36) compared to toxicity testing based on the inhibition of fluorescence from Vibrio
fischeri, which is most frequently applied in processes for removal of OPs, but is three
times less sensitive for detection of oxo-organophosphates, compared to
phosphorothionates (37).
But, AChE is not inhibited by thio-OPs, and assays such as FIA-AChE-TLS require
oxidation of thio-OPs into AChE inhibiting oxo-OPs (Fig. 4) prior to their determination.
Efficient batch mode procedures for oxidation of thio-OPs were recently developed for
the purpose of TLS and are based on chemical oxidation (38), as well as on application
of enzymes such as chploroperoxidase (CPO) isolated from marine fungi Caldariomyces
fumago (15, 16, 26, 39, 40).

8
 Malathion Malaoxon

Figure 4: Oxidation of a thio-organophosphate (Malathion) into an oxo-organophosphate

(Malaoxon) as can result from the action of P450, CPO or chemical oxidants.

CPO is a versatile enzyme with the capability of peroxidative, catalactic, halogenating,

and chlorinating activity (41), acting on large spectrum of substrates. But, from the
perspective of determination of organophosphates, the ability of CPO to induce oxygen
transfer reactions that resemble those induced by P450 monooxygenases (42) is much
more important. However, the source of oxygen has to be added to reaction medium in
the form of hydrogen peroxide (H2O2) or some other hydroperoxide (methyl- or ethyl-
hydroperoxides, tert-butyl hydroperoxide) (43, 44). CPO offers selective and quantitative
batch-mode oxidation and improvements of LODs for determination of malathion and
chlorpyrifos by 850 and 279-times, respectively (39).
The dependence of CPO activity on pH, imposes additional requirements and constrains
on experimental conditions for FIA determination of organophosphates with on-line
oxidation of thio-OPs by CPO (15, 16). Furthermore, the optimal pH is different for the
two enzymes (CPO and AChE) used in the FIA system. CPO shows highest activity in
acidic (pH = 3) while AChE in neutral (pH = 8-9) pH range. A FIA manifold with two
pumps (as shown on Fig. 3) is therefore needed and operational parameters should be
adjusted carefully to achive optimal pH for the action of AChE. Combination of 0.2 M
phosphate buffer with pH 9.1 and 0.025 M citrate buffer with pH 2.95 in 2:1 flow-rate
ratio, resulting in 0.6 mL/min cummulative flow-rate on the AChE column, and final pH
value of 7.4, provide conditions for the highest AChE activity and the highest inhibition
effect in the given FIA system.
Despite the known CPO inactivation by H2O2 (43, 44) no effect of peroxide on CPO
enzyme was observed with 0.1 M H2O2 for injections of up to 15 samples. For samples
requiring higher H2O2 concentrations due to matrix effects (i.e. sugars in fruit juices) a

9
progressively faster CPO inactivation was observed already at lower numbers of samples
(i.e. six samples at 1 mM H2O2).
By integrating the on-line oxidation performed with immobilized CPO enzyme in a FIA-
TLS system, the detection sensitivity and LODs for tested thio-OP analogues (malathion,
parathion-methyl, chlorpyrifos and diazinon) were improved. The achieved LODs were
27 μg/L, 28 μg/L, 55 μg/L and 500 μg/L for chlorpyrifos, malathion, parathion-methyl
and diazinon, respectively. Detection of unoxidized parathion-methyl and diazinon was
not possible even at 144 mg/L and 40 mg/L concentration, respectively (upper limit of
their water solubility). Equally important is the observation that with the incorporation of
the CPO oxidation step the time needed for one sample analysis was not increased in
comparison to AChE-FIA-TLS system described earlier (24, 30) and is in the range of 7
to 10 minutes per sample. This is by factor of 12 shorter in comparison to AChE assays
with chemical or enzymatic batch oxidation (45, 46).

Thermal lens microscopy for bioanalytical methods

An important step in the application of TLS detection in flow injection analysis was the
development of the thermal lens microscope (TLM) (47), which enabled several new
applications of TLS in microchemical analysis (48). This progress stems from the unique
geometry of pump and probe beams in TLM, which are aligned coaxially under the
microscope and focused with a single chromatic lens. The resulting chromatic aberration
of a few μm enables detection of analytes in microwells and microchannels, which can
not be performed on an analytical microchip by the transverse mode TLS. In addition,
microchips provide a platform for various steps of chemical analysis, such as mixing of
analytes and reagents, liquid or solid phase extraction, phase separation, concentration of
gaseous analytes, growing of cell cultures, as well as sample heating, which can be
performed on-line (48).
The versatility of microchip based TLM detection offers many other interesting
configurations for bioanalytical assays. Among those relying on TLM detection in flow, a
microchip-based microELISA for determination of interferon-γ (17), should be
mentioned. In this case the polystyrene microbeads precoated with the interferon (IFN)
antibody were placed into a microchip channel. In the first step of analysis the sample
containing IFN was introduced, and IFN was captured by the first antibody. The
injections of the second (biotinylated) anti-IFN and streptavidin-horseradish peroxidase
conjugate followed successively. Finally the substrates (H2O2 and N-ethyl-N-(2-hydroxy-
3-sulfopropyl)-3-methylaniline, sodium salt) and 4-aminoantipyrine were injected
through separate channels, and the resulting product was detected downstream by TLM.
An absolute amount of 6 pM of IFN (LOD = 0.1 ng/mL) could be detected by the
described microELISA procedure.
A similar principle was used for detection of secretory human immunoglobulin A (s-
IgA), which was initially adsorbed on polystyrene beads placed in a microchannel of a
glass chip. The secondary antibodies (anti-s-IgA) were in this case conjugated with
colloidal gold and after their interaction with s-IgA on polystyrene beads, the TLM
quantification of 1 g/mL of s-IgA was possible at 514.5 nm excitation wavelength of an
Ar-ion laser (49). Due to the 37-times reduced sample volume/sorbent surface ratio in a

10
microchip, as compared to microtiter plates in conventional ELISA tests, the antigen-
antibody reaction was completed in 10 minutes as deduced from the stabilization of TLM
signal. This represents a substantial improvement in sample throughput compared to over
15 hours required for the conventional bulk scale ELISA and enables sensitive
determination of s-IgA in saliva, where concentrations are normally on the order of 200
g/mL.
Colloidal gold conjugated antibodies of immunoglobulin G (IgG) were used for detection
of carcinoembryonic antigen (CEA) in human sera – a commonly used marker of colon
cancer (50). In this case the assay requires sequential addition of two antibodies (rabbit
anti-CEA and colloid gold conjugated IgG), following the binding of CEA to a mouse
anti-CEA antibody adsorbed on polystyrene beads. Still, the entire assay can be
completed in 35 minutes (compared to 45 h with ELISA on microtiter plates) with the
detection limit of 0.03 ng/mL CEA, which enabled assays of patient’s sera with CEA
concentrations in the range of 0.67 – 7.3 ng/mL.

Bioanalytical applications of TLS on cell cultures and single cells

Growing cell cultures in clinical, biochemical and other laboratories had already become
a routine (51). It is therefore not surprising that bioanalytical assays employing TLS have
already been developed.
Considering first the indirect observations of biomolecules and their actions, the studies
of bilirubin transport across the cellular membrane shall be discussed. For this purpose
cells of human hepatocellular liver carcinoma cell (HepG2) line were grown to
confluence in 25 cm2 flasks. The intake of bilirubin by the cells was monitored by TLS
through the decrease in its concentration in the growth media, which was clearly
observed on one minute time scale. Due to very low solubility of bilirubin under
physiological conditions (max. 50-70 nM), which is not accessible by spectrophotometry,
a novel TLS method for determination of bilirubin was developed (52). It enabled
detection of bilirubin (LOD = 1 nM) under physiological conditions, and was further
adopted for detection in FIA mode to avoid losses in sensitivity due to photolability of
bilirubin (53). Compared to batch mode TLS determination (52, 54), a 2.5-fold
improvement in LOD was achieved by FIA-TLS with carrier flow rate of 2 mL/min and
injections of 200 μL samples (substrates from cell cultures containing up to 50 nM
bilirubin, diluted by EtOH 1:1) (53). In further research of this subject, specific
antisequence antibodies for the membrane protein bilitranslocase were added to the
culture medium, and the activity of bilitranslocase (BTL) was studied in presence of
substrates for other carrier proteins in the cellular membrane (digoxin) and for BTL
(nicotinic acid). The results (52, 54) represent the first experimental evidence of bilirubin
uptake by hepatic cells under physiological conditions and have confirmed the role of
BTL as carrier protein in this process.
While the TLS measurements described above were performed off-line, a novel double
dual-beam TLS instrument was designed to observe on-line the effects of a cytotoxin (3-
alkylpyridinium polimers from a marine sponge) on phytoplankton cell culture
(Skeletonema costatum). The lysis of cultured cells (6106 – 107 lysed cells/L) was
monitored utilizing the TLS, which detected with high sensitivity (excitation at 476.6

11
nm, 100 mW) the presence of pigments released from the cell cytoplasm (mainly
carotenoids) (55).
Progress in cell culture technology and fabrication of microchips has recently enabled
growing cells on microchips (56), which enables scaling down of the experiments on cell
cultures to a single cell observation as predicted for TLM (57). Similarly to the
monitoring of pigments release from lysed phytoplankton cells, a TLM method was
developed to monitor intercellular messengers such as arachidonate (58) during its
release from the rat nerve cells, kept in a 0.1 mm deep, 1 mm wide and 10 mm long
culture chamber on a microchip. The release of arachidonate, the retrograde messenger
from the cells was stimulated by the injection of glutamate, which is a known
neurotransmitter. Arachidonate was detected by TLM at 244 nm excitation wavelength.
The detected concentration of 1.3×10-5 M (for 10-4 M glutamate) corresponded to 1.3
fmol of arachidonate released per cell (58).
By analogy to the investigation of phytoplankton cell lysis, a TLM system (532 nm
excitation wavelength) was applied to two dimensional monitoring of cytochrome c
distribution in a neuroblastoma-glioma hybrid cell cultured in the micro-flask (1 mm ×
10 mm × 0.1 mm; 1 L) fabricated in a glass microchip (59). Cytochrome c release from
mitochondria to cytosol during the apoptosis of the cell was successfully monitored with
a 1 m resolution, after addition of staurosporin, which triggered the apoptosis. The
absolute amount of cytochrome c detected in the probed volume was estimated to be
about 10 zmol.
A further important step in application of TLS to studies on single cells was achieved by
the detection and measurement of human leukocyte antigens (HLA) on the surface of a
single blood cell (60). Antigens of HLA-A, -B, and -C loci on the lymphocytes were
identified and quantified by TLM and an image of HLA-A, -B, and -C antigen
distribution on a mononuclear leukocyte was obtained (1.5 – 2 m resolution). For this
purpose anti-HLA-A, -B, and –C antibodies were labeled with colloidal gold and added
to mononuclear leukocytes for incubation (1 h at 5 C) before being assayed. The TLM
laser microscope was specially devised for measuring convex surface cells, which are 9-
12 m in diameter, and the deviation of the TLM focal point from the cell surface was
corrected by utilizing the phase of the signal.

CONCLUSIONS AND FUTURE OUTLOOK

The described examples of applications and listed references demonstrate that TLS and
TLM had found many applications in bioanalytical assays. These are not only related to
determinations of various compounds by utilizing biomolecules, but also to
investigations of various processes on cell cultures and even single cells, which could not
be studied till now, mainly due to insufficient sensitivity or inadequate spatial resolution
and response time of the existing analytical methods.

12
A substantial number of publications in the recent few years belongs to the combination
of TLM and microchip chemistry, which goes very well along with the general trends for
miniaturization and higher throughput of analytical instruments and methods. This is also
the area where most method development and application of TLS is foreseen in the near
future. This could include some of the already developed concepts of TLM and chemistry
and biological cells on microchips (48, 56) for determination of new emerging pollutants
in the environment, such as pharmaceuticals, as well as allergens in foodstuffs. A number
of new applications in biomedical research is also very likely, as already predicted (60).
For these purposes recent advances on chemi-functional membranes on microchips (12),
pressure driven flow control systems (61) and bio-microactuators (62) could be utilized.
New developments in TLS instrumentation are also foreseen primarily in direction of
miniaturization and combinations of various detection schemes such as miniaturized
thermal lens and fluorescence detection systems (63) or circular dichroism TLM (64) for
detection on microchips. Since pulsed excitation is known to be advantageous in
configurations with tightly focused pump beams and in flowing systems as well, more
applications of pulsed TLS are expected despite their incompatibility with lock-in
detection. This can be partly eliminated by modulating the laser pulses (generated at a
high repetition rate) at a lower frequency, which serves also as s reference frequency for
lock-in detection of TLS signals (65). State of the art pulsed laser sources have also the
advantage over continuous wave lasers in more compact size and better wavelength
tuning. Availability of adequate lasers however still remains one of the major obstacles
for a more widespread application of TLS. For this reason new concepts for generation of
a thermal lens shall be exploited, such as for the case of TLM on microchips, where the
rapid thermal conduction on the solid-liquid interface resulted in generation of a
temperature gradient in a microchannel (66). Such a mechanism does not require
coherence and normal radial intensity distribution of the laser beam. Therefore, other
incoherent and tunable broad-band light sources can be utilized for sensitive detection.
Photothermal spectrometry in small sub-microliter cylindrical cells has been proposed
earlier (67, 68) and could find application in chromatographic and FIA systems as well,
providing that the experimental conditions are optimized to reduce relatively long
response times, which were reported to be on the order of several hundred milliseconds,
and are not best suited for applications in flowing media.
Nonetheless, the demonstrated instrumental versatility and high sensitivity, as well as the
wide range of applications of TLS are very much likely to attract new users of this
promising technique. On the other hand, challenges of obtaining million fold sensitivity
enhancements (69) or detection of various molecules on single cells (60), should be a
good stimulation for further advancement and application of this technique.

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