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Role of non-linear optics and multiple photon absorbance in enhancing sensitivity of enzyme-based chemical agent detectors
Methods 46 (2008) 18–24
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Role of non-linear optics and multiple photon absorbance in enhancing sensitivity of enzyme-based chemical agent detectors H. James Harmon * Department of Physics, Center for Sensors and Sensor Technology, Oklahoma State University, PS 145, Stillwater, OK 74078, USA
a r t i c l e
i n f o
Article history: Accepted 22 May 2008 Available online 16 June 2008 Keywords: Porphyrin Evanescent Chemical sensor Non-linear optics Energy transfer Singlet Triplet
a b s t r a c t Real-time chemical sensors have been developed based on the binding of the analyte to monolayers of either porphyrin alone or porphyrins incorporated into the active site of enzymes. Binding of an analyte to porphyrin alone causes a redistribution of electrons in the porphyrin, altering the energy levels of the electrons which manifests as a change in the absorbance spectrum of the porphyrin. Porphyrins incorporated into the active site of enzymes such as cholinesterases are displaced when a competitive inhibitor such as nerve agents binds to the active site; this results in the porphyrin experiencing a different microenvironment than in the protein, resulting in a change in absorbance spectrum. Based on the Beer–Lambert relationship of concentration and absorbance, the limit of detection (LOD) for porphyrin-based sensors should be approximately 2 nM although LODs several orders of magnitude lower have been published. This increased sensitivity is explained as the result of multiple photon absorbance by the porphyrin and limiting self-quenching energy transfer reactions in the evanescent monolayer. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
1.2. Background
Detection of extremely low levels of a hazardous agent, be it a weapon of mass destruction, a pollutant, or an infectious agent, is desirable from many aspects; early detection and treatment of a disease is desirable as is the definition of the ‘‘safe” zone around an industrial, environmental, or terrorist incident involving harmful entities. The desire of many is to detect a single molecule of a harmful agent in a second or less, cost little, and require no training. As difficult as this is, inroads into low level detection are occurring. However, a principle of uncertainty exists. Similar to Heisenberg’s principle, the ability to correctly identify an analyte at extremely low levels is very difficult; conversely, it is easy to correctly identify a high concentration of agent/analyte. High sensitivity is obtained at the cost of low selectivity and vice-versa. This ‘‘uncertainty” trade-off dictates the design of sensors and detectors. Achieving high selectivity of analyte detection at very low levels is difficult yet this is the very core of efforts to make sensors of high sensitivity with low ‘‘false positive” rates. Enzyme-based chemical detection has been reviewed recently. This manuscript will present an explanation of the seemingly (at first glance) impossibility to detect agents at the low levels we have presented.
‘‘Nerve agents” represent a class of WMD and CWA (chemical warfare agent) which contains well-known compounds such as sarin, soman, and VX. The nerve agents share a common aspect with other compounds such as organophosphate pesticides and even some therapeutic drugs (for dementia and Alzheimers patients) in that they all bind at the active site of acetylcholinesterase (AChE) in the target. These compounds may also bind to the ‘‘pseudo-cholinesterase” or butyrylcholinesterase (BChE) present in the blood stream. Inhibition of the AChE in the central nervous system results in the tremor, shaking, convulsive, etc. symptoms of nerve agent poisoning. Detection of a chemical compound can occur either by direct measurement of a physical property of the analyte such as the presence of a chemical group as determined by infra-red spectroscopy, the charge/mass ratio in an ion mobility tube, or other techniques that measure aspects of the chemical directly. A second type of detection involves studying the effects of interaction of a ‘‘reporter” group with the analyte; this is indirect measurement since the inherent properties of the ‘‘reporter” and not the analyte are measured. The ability of a chemical compound such as a nerve agent to absorb an IR photon is small (low absorptivity) which makes identification of low levels very difficult. On the other hand, reporter molecules with distinct spectral characteristics and the ability to absorb or emit photons can be used to provide a suitable measurable response to small levels of analyte. We have chosen the latter option of detection and have chosen as our ‘‘reporter” the porphyrins and related molecules.
* Fax: +1 405 744 6811. E-mail address: [email protected] 1046-2023/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.05.003
H.J. Harmon / Methods 46 (2008) 18–24
2. Porphyrins The basic structure of a porphyrin is a tetrapyrrole macrocycle ring which can be derivatized at its perimeter; such derivatizations alter the water solubility, absorbance spectrum, and other properties of the porphyrin. However, the ability to absorb light represented by the extinction coefficient (or oscillator strength) provides the most desirable aspect of use of porphyrins and related molecules as reporter molecules. Unfortunately, a reporter may not interact well, if at all, with its analyte. Additionally, the reporter may interact with other non-related analytes in a manner that yields similar spectral changes making definite identification of the analyte difficult if not impossible. However, different analytes frequently react with porphyrins differently to yield analyte-specific spectral changes. A commonly used porphyrin is TPPS (tetraphenylsulfonate porphyrin; Fig. 1) whose visible absorbance spectrum is shown in Fig. 2. The spectrum of TPPS in the Soret (B-band) region where the extinction coefficient is 500,000A/M/cm1 is shown in Fig. 3. In the presence of benzene, the spectrum of the porphyrin is altered since the pielectrons of the benzene interact with and perturb the pi-bonds
O
O
O
S
O
of the porphyrin macrocycle. A small red-shift in the porphyrin is observed; this shift is seen more clearly if we subtract the spectrum of the porphyrin from the sample in the presence of 10 ppm benzene in water (cf. Fig. 4). This benzene plus TPPS minus TPPS difference spectrum shows a loss of absorbance (trough) at the wavelength of the parent porphyrin and an absorbance increase at the wavelength of the TPPS-benzene complex that forms; any individual porphyrin is either complexed or not and as a result, the peak minus trough absorbance difference is proportional to the number of porphyrins which have bound with the analyte. The peak wavelength is different for different analytes: 421 nm for benzene, 419 nm, for naphthalene, 434 nm for formaldehyde, etc. However, the significant half-bandwidth (approximately 10 nm) of the porphyrins and the analyte-porphyrin complexes makes the identification of analytes whose absorbance peaks are similar (±2 nm) in a mixture quite difficult. Further, several organics induce the same spectral change as naphthalene. Thus, while the extinction coefficient allows sensitive measurements of the amount of complex (and, hence, analyte) present, selectivity may be compromised due to non-specific or spectral changes at similar wavelengths upon binding of different analytes. Porphyrins have been shown to bind proteins resulting in changes in the porphyrin absorbance spectrum [1]. 3. Enzyme-based chemical agent detection
S
O
O
N H N
N H N
O
O S
O
19
S O
O Fig. 1. Structure of TPPS.
O
To provide specificity and selectivity of the response to chemical analytes and agents, we utilize the specificity of the analyte (e.g., nerve agent) for its target (cholinesterases) or any other enzyme/protein to which the agent could bind such as organophosphate hydrolase (OPH), BChE, or even carboxypeptidase; the cholinesterases and carboxypeptidase are, like trypsin, classified as serine esterases because of the presence of a catalytic serine residue in the active site (even though the active sites are not identical) and are inhibited by similar molecules. We utilize the natural receptor (the binding site of the serine esterase) of the ‘‘nerve agent” and pesticides—the enzymes they inhibit. To combine the specificity of the enzyme active site with the sensitivity of the porphyrin reporter, we have been able to verify that porphyrin, particularly monosufonatotetraphenyl porphyrin (TPPS1), can act as a competitive inhibitor of the cholinesterases (and other serine esterases). TPPS1 binds at the active site and competes with the nerve agent/pesticide/drug for the active site. The target analyte-binding enzyme is covalently bound to glass slides with an amine surface using glutaraldehyde to form a Schiff base with the amines on the enzyme surface. TPPS1 is then incorporated into the active site simply by adding TPPS1 solution to the bound enzyme surface; one porphyrin binds per enzyme [2]. TPPS (tetraphenylsulfonate porphyrin) acts in a similar manner [1].
Fig. 2. Absorbance spectrum of tetraphenylsulfonate porphyrin (TPPS).
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H.J. Harmon / Methods 46 (2008) 18–24
Fig. 3. Absorbance spectrum of 3 lM TPPS in water (solid curve) and in the presence of 10 ppm benzene in water (dashed line).
detection system. If the detector measures the primary product or the product of a secondary coupled reaction (such as with a peroxidase) reaction at, say, a 1 lM threshold, then the enzyme must turnover until it has generated lM product. The sensors developed at Oklahoma State University consist of a single monolayer of enzyme or porphyrin covalently linked to the surface. What is measured is only the binding of the analyte to the surface or the protein and not the catalytic activity or turnover of the enzyme; the rate of binding, unlike the rate of catalysis, is not a function of the number of enzymes or receptor sites on the surface. The fraction of the enzymes/receptors bound is defined by the association and dissociation constants (or Michaelis–Menten constant for enzymes). Binding an analyte to this monolayer is not diffusion-limited as it would be if a thicker or ‘‘bulk” medium were used. In a multilayer surface, movement of the analyte to the enzyme or porphyrin is time-dependent with longer times needed for progressively thicker samples; typically the effect observed changes exponentially with time as would be expected from laws of diffusion. Exponential absorbance changes in our systems are not observed over time. 5. Discussion—the problem–why are evanescent porphyrinenzyme-based sensors so sensitive?
Fig. 4. Difference spectra (TPPS + benzene minus TPPS) at different benzene concentrations. The depth and height of the 413 nm trough and 419 nm peak increase with increasing concentrations of benzene as TPPS forms increasing amount of TPPS-benzene complex absorbing at 419 nm.
Upon binding of a cholinesterase inhibitor, the porphyrin is displaced from the active site. Since the electron distribution dictated by the microenvironment around the porphyrin will be different when TPPS1 is in the active site compared to when it is removed from the site, we expect that the absorbance spectrum of the porphyrin will change and that the magnitude of the absorbance intensity difference will reflect the amount of porphyrin displaced and hence the amount of agent present [3,4]. This is indeed the case and has been published previously for the detection of glucose using glucose oxidase [5], carbonic anhydride for CO2 [6], OPH (organophosphate hydrolase) to determine organophosphate pesticides such as diazinon or paraoxon [7] and cholinesterase inhibitors such as galanthamine, eserine, or sarin using serine esterases such as AChE [3,4,8,9], BChE [4,10], or carboxypeptidase [11]. This technique can be used, theoretically, for almost any enzyme where a colorimetric molecule can be found to serve as a competitive inhibitor of that enzyme [8,9,11–14]. The enzyme provides the substrate/inhibitor specificity and the large extinction coefficient of the colorimetric agent provides sensitivity of the measurement. This technique has allowed the measurement of the pesticide paraoxon down to seven parts per trillion in water using organophosphate hydrolase as the target enzyme [7]. 4. Speed of detection Many procedures of enzyme-based detection of chemical and biological agents require the measurement of kinetics of an enzyme prior to and following exposure to the agent. This can involve several minutes time for each measurement. This type of detection measurement is frequently necessitated by the insensitivity of the
The sensitivity of optical detection of analytes by absorbance spectroscopy is limited. Many molecules (if not most) are colorless in the visible light region where optical measurements are easiest and cost-effective since UV-producing lamps and quartz optics are not required. Thus, the compound must be detected by use of a ‘‘reporter” molecule that interacts with the analyte and changes its spectral characteristics. Reporter molecules typically have high extinction coefficients and thus are brightly colored. Assuming that a reporter molecule can be found that interacts with the analyte to alter its spectrum, we are limited in detection limits by the physical constant of the extinction coefficient. The amount of light absorbed is directly linearly related to the concentration of analyte present by the Beer–Lambert Law:
A ¼ abc
ð1Þ
where a is the extinction coefficient, b is the pathlength, and c is the concentration. Let us use a porphyrin as our example since it has an extremely large extinction coefficient; for this example we will assume that the value of a = 500,000 A/M/cm path. If we assume that our optical device can measure reliably down to 0.001 A with S/ N = 3, then
0:001 A ¼ ð500; 000=M=cmÞð1 cmÞc; c ¼ 2 109 M or 2 nM
ð2Þ
Since the detection limits such as PEL, LOD, and IDHL are frequently listed as ppm, ppb, etc., this concentration is related to the molar concentration by the molecular or formula weight. Thus, for sarin (MW = 140 g/mol), the 2 nM is 280 109 g/L, 0.28 lg/L or 0.28 ppb or 280 ppt (parts per trillion). For paraoxon (MW = 275.2 g/mol), 2 nM is 0.55 lg/L or 0.55 ppb or 550 ppt; yet we have recorded absorbance changes at the 7 ppt level with a S/ N ratio of 3 [7]. The paraoxon detection level (LOD) is approximately 79 times less than the possible detection limit defines by linear optics (Beer–Lambert). Given that direct detection and measurement, even by a highly absorbing reporter, can only detect to the hundreds of parts per trillion, the question remains of how to achieve lower detection levels. Detection at less than 10 ppt paraoxon and 100 ppt sarin using porphyrins incorporated into organophosphate hydrolase (OPH) and acetylcholinesterase (AChE), respectively, with have been reported [7,8]. Since the extinction coefficient of the porphyrins is on the order of 500,000 A/M/cm, this does not seem possible.
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H.J. Harmon / Methods 46 (2008) 18–24
Soret
Increased absorbance can be attained several different ways. First, while the extinction coefficient of free unliganded porphyrin may be 500,000 A/M/cm, this value may increase when the porphyrin is bound into a different environment in the active site of the enzyme. Substantial changes in oscillator strength (extinction coefficient and absorbance intensity) can result from perturbations or distortions of the porphyrin structure when the porphyrin binds the protein. Further, the measurements published showing 7– 100 ppt detection levels utilize the peak-vs.-trough absorbance difference of the post-minus-pre-exposure spectra; the loss of absorbance of the porphyrin (trough) occurs as the unliganded/unbound porphyrin is formed; this in effect doubles extinction coefficient of the porphyrin since the extinctions of the bound and unbound porphyrin are similar. However, the complex of TPPS1 with AChE results in an absorbance peak at 442 nm while the peak of the TPPS1–BChE complex is at 429 nm, indicating that the porphyrin is in a much different environment (the active sites of the two proteins are different although each has a serine at the bottom). Unfortunately, the trough and the peaks may be of similar intensity [4], indicating that the oscillator strength (extinction coefficient) of the enzyme-bound porphyrin is similar to that of the porphyrin in water even though its absorbance spectrum is altered. Unfortunately, this does not increase the absorbance values.
Q-Bands
5.1. Absorbance by excited states: multiple photon absorbance Fig. 5. Energy level diagram of absorbance and emission dynamics of TPPS in water. Dashed downward lines represent non-radiative decay from singlet excited states; heavy upward lines represent absorbance of the singlet and triplet excited states.
1.5
ABSORBANCE
The absorbance of a photon in the B- or Soret band of the porphyrin (400–450 nm) increases the energy of a ‘‘ground state” electron of the porphyrin macrocycle resulting in the formation of a singlet excited state; the first singlet S1 excited state absorption coefficient for tetraphenyl porphyrin (H2TPP) is 2.35-fold greater than that of the Soret ‘‘ground” or unexcited state absorption coefficient [15]. Indeed, the absorbance of yet a third photon has been demonstrated in porphyrin [16] to generate excited singlet state Sn at an even higher energy level, resulting in an even larger apparent absorbance by a single porphyrin; three photons are absorbed in the Soret region with the second and third experiencing greater absorptivity (cf. Fig. 5). Indeed, these three photons could (in the case of H2TPP) show the same absorptivity as four photons (of ground state absorbance of photon). In porphyrins, the singlet excited state can undergo intersystem crossing (IC) to a triplet state which is also capable of absorbing two photons; the spectrum of the triplet excited state is similar to that of the ground and singlet excited states [17] although the absorption coefficient of the triplet state of tetraphenylporphyrin (H2TPP) is also reported 1.5 times that of the singlet ground state absorptivity [15]. The net result is that a single photon absorbance can result in at least three additional photons being absorbed by a single molecule [18]. Reindl and Penzkofer [19] reported triplet to singlet IC as well as singlet to triplet IC; crossing from the second excited triplet state T2 or higher state to the S1 or higher state returns the electron to a condition where it can again absorb a photon. The transfer from different states can proceed due to the number of radiationless transitions that dissipate the energy and allow for multiple photon absorbance by a single porphyrin molecule. As seen in Fig. 6, excitation of TPPS at 413 nm (the Soret or Bband) in the ground state results in the formation of excited states which can absorb a photon at around 416–418 nm and with comparable intensity to the ground state. The ratio of excited state absorptivity to ground state absorptivity is different for different porphyrin structures [15]; H2TPP has a value of 1.5 [15] while we observe H2TPPS to have a ratio of unity in the Soret band absorbance range. Gratz and Penzkofer [16] indicate that essentially the same fluorescence spectra are obtained from excitation at the B-band
Not Illuminated
Illuminated at 413 nm
415 nm peak
419 nm peak
1
.5
0 410
420
430
440
WAVELENGTH (nm) Fig. 6. Ground state (solid line) and excited singlet state (413 nm excitation) spectra (dashed line) of 3 lM TPPS in water. Spectra recorded with CARY 4E UV–vis spectrophotometer.
(405 nm) and the Qa(1) band (543 nm) of H2-tetraphenylporphyrin (H2TPP); in other words, excitation at the B-band decays to the Qband before intersystem crossing (IC) to the T1 triplet state; similar results were obtained by Karotki et al. [20]. Since the excited state of the Qa band is expected to be similar if achieved by direct excitation or decay from the B-band, it may be possible for excited Q to absorb a photon and enter a higher excited state (cf. Fig. 5). In the complete ‘‘cycle” from excitation of and removal of an electron from the ground state following absorbance of a ‘‘Soret” photon (all of our detection/sensor measurements utilize 413 nm light with a half-bandwidth of approximately 8–10 nm) until the electron ultimately returns to the ‘‘ground state”, at least five photons can be absorbed by the same single porphyrin. Thus, alteration of one porphyrin has the same absorbance intensity change as if five had been affected; this is essence an ‘‘amplification” of the detection event. A single porphyrin affected exhibits the same
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H.J. Harmon / Methods 46 (2008) 18–24
absorbance change of several porphyrins being affected; detection of 56 ppt or less is the same as for 280 ppt monomeric porphyrin absorbing one and only one photon assuming all excited states absorb as strongly as the ground state, which they do not. Factoring the increased absorptivity of the singlet and triplet states (as seen in H2TPP), a single porphyrin could absorb up to seven photons, the same as seven porphyrins each absorbing a single photon in the ground state; 40 ppt shows the same absorbance change as 280 ppt. 5.2. Upconversion is evidence of multiple photon absorbance Upconversion is a result of multiple photon absorbance when the excited state formed by absorbance of 2- (or 3) photons decays to ground state (or near ground state levels) by emitting a photon of greater energy (shorter wavelength) than either of the absorbed photons (the two photons absorbed need not be the same wavelength). Alternatively, the loss of energy may occur by a radiationless transition where no photon is emitted). The emission of a shorter wavelength photon is evidence that multiple photon absorbance has occurred. Extensively studied in Zn-porphyrins [21–23], the upconversion effect is also observed in non-metalloporphyrins [21,24–28]. Table 1 indicates the emitted upconverted photon wavelengths for each excitation wavelength of the Q- and near infra-red regions of H2TPPS at pH 8.6. The wavelengths in the 300–360 nm region are similar to the Nand L-UV absorbance bands of porphyrins. Similarly, the emissions at 642 and 704 nm correspond to an emitted photon as the electron moves from the T1 triplet excited state to the ground state. While we did not observe emission of a photon from 636 nm excitation of TPPS, multiple photon absorption resulting from absorbance of 700–1300 nm wavelength photons has been observed in other porphyrins [28]. This indicates that non-metallated porphyrin can also absorb multiple photons. Multiple photon absorption is typically performed with lasers at sufficient power to deliver a second photon during the lifetime of the excited state. The lifetime of the S1 singlet excited states in TPP is about 20 ns while the fluorescence lifetime is 9 ns; the lifetime if the T1 triplet excited state is 800 ns [15]. This allows continuous illumination at intensities far less that those of lasers to effect multiple photon absorbance. Furthermore, the absorbance crosssection (corresponds to the extinction coefficient) of the excited state is greater that of the ground state absorbance [15]; Drobizhev et al. [24] indicate weak ground state absorbance of the Q- and near IR bands but strong excited state absorbance at the same wavelengths (as the ground state). As a result, multiple photon absorbances can be observed using the continuous output light sources normally found in commercial spectrophotometers and fluorometers. However, multiple absorbance by excited states showing greater absorptivity does not explain the detection of the absorbance change corresponding to 7 ppt analyte [7] and thus 7 ppt porphyrin. Another mechanism may be likely involved.
Table 1 Emitted/fluoresced photons by TPPS as a result of multiple photon absorbance at various wavelengths Excitation wavelength (nm)
Emitted upconverted photon wavelength (nm)
517 554 582 636 700 750
305 321 355, 550 — 642 642, 704
5.3. Structure-induced absorbance changes The role of molecular structure in multiple photon absorbance should be considered. Changes in the symmetry or structure, such as a distortion, of a porphyrin can affect its absorbance. Two-photon absorbance in the Q-bands can be ‘‘forbidden”; the low extinction coefficient or absorbance cross-section is a result of a ‘‘forbidden (or non-favorable) transition”; this prohibition can be removed if the symmetry of the molecule (in this case, a porphyrin) increases either due to parity changes, spin interactions, or molecular structural distortions. In our research using monosulfonatotetraphenyl porphyrin (TPPS1) non-covalently bound into the active site of enzymes [2–12], small shifts in the absorbance maximum of the porphyrin occur upon binding to the enzyme. The shift is different for each different enzyme (cf. Table 2 below) since the microenvironment and the change in structure (distortion) of the porphyrin will be different in each enzyme active site; the wavelength maximum of non-bound TPPS1 is 405 nm. It is likely that the 7–21 nm red-shift of absorbance wavelength maxima reflects a change in the symmetry or structure of the porphyrin; this shift could increase the two-photon absorbance. Harmon [1] reported both the bathochromic (to longer wavelength) and hypsochromic (to shorter wavelengths) of tetraphenylsulfonate porphyrin (TPPS) on binding to different proteins. The phenyl rings on the porphyrin likely change their position and orientation to the porphyrin macrocycle plane, increasing the coupling of electronic and vibronic wavefunctions, contributing to an increase in multiphoton absorbance [28]. Karotki et al. [20] explain that a decrease in electronic symmetry can occur due to solvent effects (as would likely occur when the TPPS1 molecule leaves the active site and enters the aqueous medium) resulting in a change in two-photon absorbance cross-section. Similarly, a change in the dipole moment of the porphyrin as would occur as it associates with and dissociates from the enzyme will affect symmetry and the two-photon absorbance. Drobishev et al. [27] indicate drastic differences in two-photon absorbance in porphyrins with different electronaccepting groups at the periphery; this suggests that a change in electron distribution of the porphyrin will result in a change in two-photon absorbance intensity as well as alterations in the ground state spectrum. Thus, it is likely that changes in absorptivity in excited state absorbance may be the result of changes in porphyrin structure and electron distribution but will not on its own increase the sensitivity of detection beyond what has been presented thus far. 5.4. Concentration dependence of non-linearity of evanescent absorbance The detector response frequently is not linear with the concentration of the analyte being detected; instead, the change in absorbance may be proportional to the log of the analyte concentration (e.g., Refs. [3,4,10]). The apparent absorbance changes are significantly greater at lower concentrations than at higher concentrations. This is another ‘‘deviation” from the Beer–Lambert absorbance relationship; at higher concentrations of analyte, the absorbance change decreases. Such a deviation has been observed
Table 2 Wavelength maxima of TPPS1 incorporated into the active site of different enzymes Enzyme
Wavelength maximum of the enzyme–TPPS1 complex (nm)
AChE (acetylcholine esterase) BChE (butyrylcholine esterase) OPH (organophosphate hydrolase) CBPA (carboxypeptidase A)
426 421 412 423
H.J. Harmon / Methods 46 (2008) 18–24
by others [29–35] performing evanescent absorbance measurements using planar waveguides; the apparent evanescent absorbance exceeds bulk solution-based (e.g., with 1 cm cuvettes) absorbance of the same material. The basis of this has been addressed [29,36] with evanescent absorbance in coated optical fibers. Ruddy et al. [29] showed that the evanescent absorbance of a fixed analyte concentration varies linearly with the interaction length; they also indicated that at low analyte concentrations, the evanescent absorbance can be over 10 times greater than the bulk absorbance effect in 1 cm cuvettes. The net effect from these studies is that with low concentrations of analyte absorbed onto a planar waveguide, the evanescent absorbance can exceed bulk absorbance of the same material over 10-fold. This allows for accurate measurement of low analyte concentrations; the apparent measured absorbance for a given analyte concentration is greater than expected from the bulk Beer–Lambert relationship, as we have reported previously. 5.5. The role of self-quenching in evanescent thin film sensor response The enhancement of absorbance in evanescent measurement of thin films may also be due to the limitation of ‘‘quenching” reactions of the porphyrin. In solution, the porphyrin will absorb a photon in the ground state and enter an excited state; the excited state can decay via a radiationless transition within the porphyrin and/ or absorb a photon and move to a higher energy state (from the singlet or triplet state) or possibly become ‘‘self-quenched” where the energy of the excited state is transferred to another porphyrin molecule during a collision in solution. Thus, the absorbance in solution is limited by the number of collisions with adjacent molecules and may contribute to the non-linear deviation from Beer’s Law commonly observed (absorbance decreases at higher porphyrin concentrations); the deviation from linearity is also attributed to formation of dimers or aggregates. In a thin film of immobilized porphyrins or porphyrins surrounded by protein (as in the case of TPPS1 bound in the active site), the ability to freely collide with and transfer energy to adjacent porphyrins is severely limited. If intramolecular energy transfer cannot occur, the possibility for multiple photon absorbance to occur increases, enhancing the apparent absorbance intensity as explained earlier. We would expect that at higher density of porphyrins on the thin film, the possibility of energy transfer to an adjacent molecule will increase, limiting the photon absorbance to exciting a ground state electron only. In solution, porphyrins can interact and transfer energy in all directions of space while in the thin film interaction is limited to the plane of the surface (monolayer of absorber) and is facilitated by higher loading of the surface resulting in closer proximity of the energy acceptor molecules. The observed effect would be that the evanescent absorbance would increase with the amount of bound material until which time the excited molecules are able to freely interact with neighboring molecules, returning the donor molecule to the (single-photon-absorbing) ground state and exciting the acceptor into an excited state without benefit of a further (multiple) photon absorbance; the amount of light absorbed is thus less than if there were no interaction and the slope of absorbance vs. concentration curve would decrease with increasing analyte concentration, as observed [29]. Lakowicz and Weber [37] observed that I quenching of ethidium bromide fluorescence when bound to double helical DNA is 1/30 as effective as when the dye is in solution. They concluded that the quenching mechanism involves a quenching radius equivalent to the collisional radius regardless of the medium. Suh and Chaires [38] performed similar experiments and concluded that the fluorescent donor and acceptor (quencher) must be within a certain distance of each other for energy transfer to occur (this
23
can be a short distance in free solution but may be large in immobilized systems. Maniara et al. [39] studied phosphorescence of ANS (1-anilinonaphthalene-8-sulfonate) and TNS [(2-(p-toluidinyl)naphthalene-6-sulfonate] in solution and bound to bovine serum albumin (BSA) and, on the observation that phosphorescence is not observed in solution, concluded that molecules undergo rapid non-radiative decay from the singlet when free in solution. Since this competes with triplet formation, absorbance of photons in the singlet and triplet states will not occur; hence, enhanced absorbance will not be seen. Turro et al. [40] observed that the phosphorescence lifetimes of 1-bromonaphthalene and 1-chloronaphthalene increase when bound to cyclodextrin; the net result is that immobilization increases the lifetime of excited states, allowing a greater probability for photon absorbance by the excited state. Similarly, quenching of the sex steroid d1,3,5(10),6,8-estrapentaene-3,17b-diol [hydroequilenin (DHE)] by acrylamide decreased when the steroid was bound to rabbit sex steroid binding protein [41]. Vaughn and Weber [42] observed that the fluorescence of pyrenebutyric acid bound to BSA, hemoglobin, and polylysine is not affected by the concentration of oxygen, unlike when the fluor is in free solution. The rate of quenching of the excited state of the fluorescent molecules by collision with a quencher molecule is directly proportional to D, the summed diffusion coefficients of the fluor and quencher, N0 the number of molecules per volume, and a, the encounter distance (sum of molecular radii). The rate of quenching is expressed by the following expression:
k ¼ cð4paDN0 Þ
ð3Þ
with the factor of 4p indicative of the 3-dimensional aspect of the quencher affecting the fluorophore where c is the quenching efficiency. However, if one or both of the molecules are immobilized, the diffusion coefficient is drastically decreased. Thus the multiple photon absorbance and enhanced evanescent absorbance are related to each other at low sensor indicator (e.g., the porphyrin) concentrations where the effective distance between fluorescent donor and acceptor exceeds the effective transfer distance and where immobilization of the molecules restricts the ability to encounter each other and transfer energy. 6. Conclusion This report describes the selectivity and sensitivity of chemical and biological detectors by the use of a colorimetric indicator (porphyrin) which is incorporated into the active site of specific enzymes and incorporated into the biomimetic or naturallyoccurring binding/receptor site for the biologicals. In each case, binding of the porphyrin or its complex changes the electron distribution of the porphyrin which alters the absorbance spectrum of the porphyrin. The change in absorbance intensity is frequently non-linearly proportional to the amount of agent/analyte present and bound. On the basis of known and published extinction coefficients of the porphyrin, the limit of detection derived from Beer’s Law is approximately 2 nM although analytes can be detected at far lower levels. This apparent discrepancy between the expected and observed values is most likely the result of multiple photon absorbance by the porphyrin in the ground state and in multiple singlet and excited states such that a single porphyrin molecule can absorb multiple photons during the ‘‘cycle” of photon absorbance in the grounds state until the electron returns to or near the ground state levels. The outcome is ‘‘amplification” of the binding such that binding of a single analyte molecule to a single porphyrin results in the absorbance of several photons; the absorbance intensity mimics the effect of several porphyrins each absorbing a photon.
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H.J. Harmon / Methods 46 (2008) 18–24
This is a deviation from the Beer–Lambert relationship. The more typical deviation form the relationship is observed when porphyrins aggregate; the apparent extinction coefficient decreases with increasing porphyrin concentration as a result of aggregation, each aggregate absorbing light as if it were a single porphyrin. In the present instance, the deviation from the Beer–Lambert relationship is opposite: a single porphyrin absorbs light as if it were multiple single-photon-absorbing porphyrins; this is a type of non-linear optics. This optical absorbance amplification is consistent with the non-linear optical properties of porphyrins and related molecules. The use of multiple photon absorbance can significantly increase the sensitivity of optical absorbance-based detectors. The absorbance of multiple photons coupled with the enhanced absorbance in evanescent measurements because of minimization of intramolecular energy transfer and energy quenching combine to give increased apparent measurable absorbance in our evanescent sensors. The combined effect is expected to be multiplicative; thus measurements of concentrations 10- to 100-fold less than expected in bulk solution measurements (using the same extinction/attenuation coefficients) can be expected depending on the number of photons absorbed by the porphyrin. Such an expectation is consistent with the observed results where the observed absorbance change is much greater than that predicted from simple Beer–Lambert type absorbance-concentration relationships. The nature of enzyme-based sensors has been reviewed recently [9,12]. The function of these sensors is a wondrous combination of chemical interactions, biochemical enzymatic specificities, and non-linear optical absorbance. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
H.J. Harmon, Biosens. Bioelectron. 16 (2001) 1035–1041. B.J. White, H.J. Harmon, Biosens. Bioelectron. 17 (2002) 463–469. B.J. White, J.A. Legako, H.J. Harmon, Biosens. Bioelectron. 17 (2002) 361–366. B.J. White, J.A. Legako, H.J. Harmon, Sens. Actuators B 89 (2003) 107–111. B.J. White, H.J. Harmon, Biochem. Biophys. Res. Commun. 296 (2002) 1069– 1071. B.J. White, H.J. Harmon, Sens. Lett. 3 (2005) 59–65. B.J. White, H.J. Harmon, Biosens. Bioelectron. 20 (2005) 1977–1983. B.J. White, H.J. Harmon, Sens. Lett. 3 (2005) 36–41. B.J. White, H.J. Harmon, Encyclopedia of Sens. 5 (2006) 1–24.
[10] B.J. White, J.A. Legako, H.J. Harmon, Sens. Actuators B 91 (2003) 138–142. [11] B.J. White, A. Oliver, H.J. Harmon, in: M.K. Ram, V.R. Bhethanabotla (Eds.), Sensors for Chemical and Biological Applications, Taylor and Francis, London, 2007. [12] B.J. White, H.J. Harmon, in: J.G. Reynolds, G.E. Lawson (Eds.), Polymers and Materials for Anti-Terrorism and Homeland Defense, American Chemical Society, Washington, DC, 2007. [13] B.J. White, J.A. Legako, H.J. Harmon, Biosens. Bioelectron. 18 (2002) 729– 734. [14] H.J. Harmon, US Patent #6, 821, 738, (2004). [15] K.J. McEwan, G. Bourhill, J.M. Robertson, H.L. Anderson, J. Nonlinear Opt. Mat. 9 (2000) 451–468. [16] H. Gratz, A. Penzkofer, Chem. Phys. 254 (2000) 363–374. [17] I. Cohanoschi, L. Echeverria, F.E. Hernandez, Chem. Phys. Lett. 419 (2006) 33– 36. [18] G.P. Das, R. Vaia, A.T. Yeates, D.S. Dudis, Synth. Met. 116 (2001) 281–283. [19] S. Reindl, A. Penzkofer, Chem. Phys. 211 (1996) 431–439. [20] A. Karotki, M. Drobizhev, M. Kruk, C. Spangler, E. Nickel, N. Mamardashvili, A. Rebane, J. Opt. Soc. Am. B 20 (2003) 321–332. [21] W. Blau, H. Byrne, W.M. Dennis, Opt. Commun. 56 (1985) 25–29. [22] S. Tamura, N. Kishii, N. Asai, J. Seto, J. Lumin. 56 (1993) 93–97. [23] S. Machida, O. Kyono, K. Horie, R. Matsuno, T. Yamashita, Polymer 35 (1994) 3915–3919. [24] M. Drobizhev, A. Karotki, A. Rebane, C.W. Spangler, Opt. Lett. 26 (2001) 1081– 1083. [25] A. Karotki, M. Kruk, M. Drobizhev, A. Rebane, E. Nickel, C.W. Spangler, IEEE J. Sel. Top. Quant. Electron. 7 (2001) 971–975. [26] M. Drobizhev, A. Karotki, M. Kruk, A. Krivokapic, H.L. Anderson, A. Rebane, Chem. Phys. Lett. 370 (2003) 690–699. [27] M. Drobizhev, A. Karotki, M. Kruk, N. Zh. Mamardashvili, A. Rebane, Chem. Phys. Lett. 361 (2002) 504–512. [28] M. Kruk, A. Karotki, M. Drobizhev, V. Kuzmitsky, V. Gael, A. Rebane, J. Lumin. 105 (2003) 45–55. [29] V. Ruddy, B.D. MacCraith, J.A. Murphy, J. Appl. Phys. 67 (1990) 6070–6074. [30] M.D. DeGrandpre, L.W. Burgess, Anal. Chem. 60 (1988) 2582–2586. [31] M.D. DeGrandpre, L.W. Burgess, Appl. Spectrosc. 44 (1990) 273–279. [32] P.H. Paul, G. Kychakoff, Appl. Phys. Lett. 51 (1987) 12–14. [33] L. Yang, S.S. Saaverda, Anal. Chem. 67 (1995) 1307–1314. [34] L.C. Shriver-Lake, K.A. Breslin, P.T. Charles, D.W. Conrad, J.P. Golden, F.S. Ligler, Anal. Chem. 34 (1995) 2431–2435. [35] H.S. Haddock, P.M. Shankar, R. Mutharasan, Sens. Actuators B 88 (2003) 67–74. [36] V. Ruddy, Opt. Eng. 33 (1994) 3891–3894. [37] J.R. Lakowicz, G. Weber, Biochem. Bioinorg. 12 (1973) 4161–4170. [38] D. Suh, J.B. Chaires, Bioorg. Med. Chem. 3 (1995) 723–728. [39] G. Maniara, J.M. Vanderkooi, D.C. Bloomgarden, H. Koloczek, Photochem. Photobiol. 47 (1988) 207–208. [40] N.J. Turro, J.D. Bolt, Y. Kuroda, I. Tabushi, Photochem. Photobiol. 35 (1982) 69– 72. [41] E. Casali, P.H. Petra, J.B.A. Ross, Biochemistry 29 (1990) 9334–9343. [42] W.M. Vaughn, G. Weber, Biochemistry 9 (1970) 464–473.
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Role of non-linear optics and multiple photon absorbance in enhancing sensitivity of enzyme-based chemical agent detectors H. James Harmon * Department of Physics, Center for Sensors and Sensor Technology, Oklahoma State University, PS 145, Stillwater, OK 74078, USA
a r t i c l e
i n f o
Article history: Accepted 22 May 2008 Available online 16 June 2008 Keywords: Porphyrin Evanescent Chemical sensor Non-linear optics Energy transfer Singlet Triplet
a b s t r a c t Real-time chemical sensors have been developed based on the binding of the analyte to monolayers of either porphyrin alone or porphyrins incorporated into the active site of enzymes. Binding of an analyte to porphyrin alone causes a redistribution of electrons in the porphyrin, altering the energy levels of the electrons which manifests as a change in the absorbance spectrum of the porphyrin. Porphyrins incorporated into the active site of enzymes such as cholinesterases are displaced when a competitive inhibitor such as nerve agents binds to the active site; this results in the porphyrin experiencing a different microenvironment than in the protein, resulting in a change in absorbance spectrum. Based on the Beer–Lambert relationship of concentration and absorbance, the limit of detection (LOD) for porphyrin-based sensors should be approximately 2 nM although LODs several orders of magnitude lower have been published. This increased sensitivity is explained as the result of multiple photon absorbance by the porphyrin and limiting self-quenching energy transfer reactions in the evanescent monolayer. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
1.2. Background
Detection of extremely low levels of a hazardous agent, be it a weapon of mass destruction, a pollutant, or an infectious agent, is desirable from many aspects; early detection and treatment of a disease is desirable as is the definition of the ‘‘safe” zone around an industrial, environmental, or terrorist incident involving harmful entities. The desire of many is to detect a single molecule of a harmful agent in a second or less, cost little, and require no training. As difficult as this is, inroads into low level detection are occurring. However, a principle of uncertainty exists. Similar to Heisenberg’s principle, the ability to correctly identify an analyte at extremely low levels is very difficult; conversely, it is easy to correctly identify a high concentration of agent/analyte. High sensitivity is obtained at the cost of low selectivity and vice-versa. This ‘‘uncertainty” trade-off dictates the design of sensors and detectors. Achieving high selectivity of analyte detection at very low levels is difficult yet this is the very core of efforts to make sensors of high sensitivity with low ‘‘false positive” rates. Enzyme-based chemical detection has been reviewed recently. This manuscript will present an explanation of the seemingly (at first glance) impossibility to detect agents at the low levels we have presented.
‘‘Nerve agents” represent a class of WMD and CWA (chemical warfare agent) which contains well-known compounds such as sarin, soman, and VX. The nerve agents share a common aspect with other compounds such as organophosphate pesticides and even some therapeutic drugs (for dementia and Alzheimers patients) in that they all bind at the active site of acetylcholinesterase (AChE) in the target. These compounds may also bind to the ‘‘pseudo-cholinesterase” or butyrylcholinesterase (BChE) present in the blood stream. Inhibition of the AChE in the central nervous system results in the tremor, shaking, convulsive, etc. symptoms of nerve agent poisoning. Detection of a chemical compound can occur either by direct measurement of a physical property of the analyte such as the presence of a chemical group as determined by infra-red spectroscopy, the charge/mass ratio in an ion mobility tube, or other techniques that measure aspects of the chemical directly. A second type of detection involves studying the effects of interaction of a ‘‘reporter” group with the analyte; this is indirect measurement since the inherent properties of the ‘‘reporter” and not the analyte are measured. The ability of a chemical compound such as a nerve agent to absorb an IR photon is small (low absorptivity) which makes identification of low levels very difficult. On the other hand, reporter molecules with distinct spectral characteristics and the ability to absorb or emit photons can be used to provide a suitable measurable response to small levels of analyte. We have chosen the latter option of detection and have chosen as our ‘‘reporter” the porphyrins and related molecules.
* Fax: +1 405 744 6811. E-mail address: [email protected] 1046-2023/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.05.003
H.J. Harmon / Methods 46 (2008) 18–24
2. Porphyrins The basic structure of a porphyrin is a tetrapyrrole macrocycle ring which can be derivatized at its perimeter; such derivatizations alter the water solubility, absorbance spectrum, and other properties of the porphyrin. However, the ability to absorb light represented by the extinction coefficient (or oscillator strength) provides the most desirable aspect of use of porphyrins and related molecules as reporter molecules. Unfortunately, a reporter may not interact well, if at all, with its analyte. Additionally, the reporter may interact with other non-related analytes in a manner that yields similar spectral changes making definite identification of the analyte difficult if not impossible. However, different analytes frequently react with porphyrins differently to yield analyte-specific spectral changes. A commonly used porphyrin is TPPS (tetraphenylsulfonate porphyrin; Fig. 1) whose visible absorbance spectrum is shown in Fig. 2. The spectrum of TPPS in the Soret (B-band) region where the extinction coefficient is 500,000A/M/cm1 is shown in Fig. 3. In the presence of benzene, the spectrum of the porphyrin is altered since the pielectrons of the benzene interact with and perturb the pi-bonds
O
O
O
S
O
of the porphyrin macrocycle. A small red-shift in the porphyrin is observed; this shift is seen more clearly if we subtract the spectrum of the porphyrin from the sample in the presence of 10 ppm benzene in water (cf. Fig. 4). This benzene plus TPPS minus TPPS difference spectrum shows a loss of absorbance (trough) at the wavelength of the parent porphyrin and an absorbance increase at the wavelength of the TPPS-benzene complex that forms; any individual porphyrin is either complexed or not and as a result, the peak minus trough absorbance difference is proportional to the number of porphyrins which have bound with the analyte. The peak wavelength is different for different analytes: 421 nm for benzene, 419 nm, for naphthalene, 434 nm for formaldehyde, etc. However, the significant half-bandwidth (approximately 10 nm) of the porphyrins and the analyte-porphyrin complexes makes the identification of analytes whose absorbance peaks are similar (±2 nm) in a mixture quite difficult. Further, several organics induce the same spectral change as naphthalene. Thus, while the extinction coefficient allows sensitive measurements of the amount of complex (and, hence, analyte) present, selectivity may be compromised due to non-specific or spectral changes at similar wavelengths upon binding of different analytes. Porphyrins have been shown to bind proteins resulting in changes in the porphyrin absorbance spectrum [1]. 3. Enzyme-based chemical agent detection
S
O
O
N H N
N H N
O
O S
O
19
S O
O Fig. 1. Structure of TPPS.
O
To provide specificity and selectivity of the response to chemical analytes and agents, we utilize the specificity of the analyte (e.g., nerve agent) for its target (cholinesterases) or any other enzyme/protein to which the agent could bind such as organophosphate hydrolase (OPH), BChE, or even carboxypeptidase; the cholinesterases and carboxypeptidase are, like trypsin, classified as serine esterases because of the presence of a catalytic serine residue in the active site (even though the active sites are not identical) and are inhibited by similar molecules. We utilize the natural receptor (the binding site of the serine esterase) of the ‘‘nerve agent” and pesticides—the enzymes they inhibit. To combine the specificity of the enzyme active site with the sensitivity of the porphyrin reporter, we have been able to verify that porphyrin, particularly monosufonatotetraphenyl porphyrin (TPPS1), can act as a competitive inhibitor of the cholinesterases (and other serine esterases). TPPS1 binds at the active site and competes with the nerve agent/pesticide/drug for the active site. The target analyte-binding enzyme is covalently bound to glass slides with an amine surface using glutaraldehyde to form a Schiff base with the amines on the enzyme surface. TPPS1 is then incorporated into the active site simply by adding TPPS1 solution to the bound enzyme surface; one porphyrin binds per enzyme [2]. TPPS (tetraphenylsulfonate porphyrin) acts in a similar manner [1].
Fig. 2. Absorbance spectrum of tetraphenylsulfonate porphyrin (TPPS).
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H.J. Harmon / Methods 46 (2008) 18–24
Fig. 3. Absorbance spectrum of 3 lM TPPS in water (solid curve) and in the presence of 10 ppm benzene in water (dashed line).
detection system. If the detector measures the primary product or the product of a secondary coupled reaction (such as with a peroxidase) reaction at, say, a 1 lM threshold, then the enzyme must turnover until it has generated lM product. The sensors developed at Oklahoma State University consist of a single monolayer of enzyme or porphyrin covalently linked to the surface. What is measured is only the binding of the analyte to the surface or the protein and not the catalytic activity or turnover of the enzyme; the rate of binding, unlike the rate of catalysis, is not a function of the number of enzymes or receptor sites on the surface. The fraction of the enzymes/receptors bound is defined by the association and dissociation constants (or Michaelis–Menten constant for enzymes). Binding an analyte to this monolayer is not diffusion-limited as it would be if a thicker or ‘‘bulk” medium were used. In a multilayer surface, movement of the analyte to the enzyme or porphyrin is time-dependent with longer times needed for progressively thicker samples; typically the effect observed changes exponentially with time as would be expected from laws of diffusion. Exponential absorbance changes in our systems are not observed over time. 5. Discussion—the problem–why are evanescent porphyrinenzyme-based sensors so sensitive?
Fig. 4. Difference spectra (TPPS + benzene minus TPPS) at different benzene concentrations. The depth and height of the 413 nm trough and 419 nm peak increase with increasing concentrations of benzene as TPPS forms increasing amount of TPPS-benzene complex absorbing at 419 nm.
Upon binding of a cholinesterase inhibitor, the porphyrin is displaced from the active site. Since the electron distribution dictated by the microenvironment around the porphyrin will be different when TPPS1 is in the active site compared to when it is removed from the site, we expect that the absorbance spectrum of the porphyrin will change and that the magnitude of the absorbance intensity difference will reflect the amount of porphyrin displaced and hence the amount of agent present [3,4]. This is indeed the case and has been published previously for the detection of glucose using glucose oxidase [5], carbonic anhydride for CO2 [6], OPH (organophosphate hydrolase) to determine organophosphate pesticides such as diazinon or paraoxon [7] and cholinesterase inhibitors such as galanthamine, eserine, or sarin using serine esterases such as AChE [3,4,8,9], BChE [4,10], or carboxypeptidase [11]. This technique can be used, theoretically, for almost any enzyme where a colorimetric molecule can be found to serve as a competitive inhibitor of that enzyme [8,9,11–14]. The enzyme provides the substrate/inhibitor specificity and the large extinction coefficient of the colorimetric agent provides sensitivity of the measurement. This technique has allowed the measurement of the pesticide paraoxon down to seven parts per trillion in water using organophosphate hydrolase as the target enzyme [7]. 4. Speed of detection Many procedures of enzyme-based detection of chemical and biological agents require the measurement of kinetics of an enzyme prior to and following exposure to the agent. This can involve several minutes time for each measurement. This type of detection measurement is frequently necessitated by the insensitivity of the
The sensitivity of optical detection of analytes by absorbance spectroscopy is limited. Many molecules (if not most) are colorless in the visible light region where optical measurements are easiest and cost-effective since UV-producing lamps and quartz optics are not required. Thus, the compound must be detected by use of a ‘‘reporter” molecule that interacts with the analyte and changes its spectral characteristics. Reporter molecules typically have high extinction coefficients and thus are brightly colored. Assuming that a reporter molecule can be found that interacts with the analyte to alter its spectrum, we are limited in detection limits by the physical constant of the extinction coefficient. The amount of light absorbed is directly linearly related to the concentration of analyte present by the Beer–Lambert Law:
A ¼ abc
ð1Þ
where a is the extinction coefficient, b is the pathlength, and c is the concentration. Let us use a porphyrin as our example since it has an extremely large extinction coefficient; for this example we will assume that the value of a = 500,000 A/M/cm path. If we assume that our optical device can measure reliably down to 0.001 A with S/ N = 3, then
0:001 A ¼ ð500; 000=M=cmÞð1 cmÞc; c ¼ 2 109 M or 2 nM
ð2Þ
Since the detection limits such as PEL, LOD, and IDHL are frequently listed as ppm, ppb, etc., this concentration is related to the molar concentration by the molecular or formula weight. Thus, for sarin (MW = 140 g/mol), the 2 nM is 280 109 g/L, 0.28 lg/L or 0.28 ppb or 280 ppt (parts per trillion). For paraoxon (MW = 275.2 g/mol), 2 nM is 0.55 lg/L or 0.55 ppb or 550 ppt; yet we have recorded absorbance changes at the 7 ppt level with a S/ N ratio of 3 [7]. The paraoxon detection level (LOD) is approximately 79 times less than the possible detection limit defines by linear optics (Beer–Lambert). Given that direct detection and measurement, even by a highly absorbing reporter, can only detect to the hundreds of parts per trillion, the question remains of how to achieve lower detection levels. Detection at less than 10 ppt paraoxon and 100 ppt sarin using porphyrins incorporated into organophosphate hydrolase (OPH) and acetylcholinesterase (AChE), respectively, with have been reported [7,8]. Since the extinction coefficient of the porphyrins is on the order of 500,000 A/M/cm, this does not seem possible.
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H.J. Harmon / Methods 46 (2008) 18–24
Soret
Increased absorbance can be attained several different ways. First, while the extinction coefficient of free unliganded porphyrin may be 500,000 A/M/cm, this value may increase when the porphyrin is bound into a different environment in the active site of the enzyme. Substantial changes in oscillator strength (extinction coefficient and absorbance intensity) can result from perturbations or distortions of the porphyrin structure when the porphyrin binds the protein. Further, the measurements published showing 7– 100 ppt detection levels utilize the peak-vs.-trough absorbance difference of the post-minus-pre-exposure spectra; the loss of absorbance of the porphyrin (trough) occurs as the unliganded/unbound porphyrin is formed; this in effect doubles extinction coefficient of the porphyrin since the extinctions of the bound and unbound porphyrin are similar. However, the complex of TPPS1 with AChE results in an absorbance peak at 442 nm while the peak of the TPPS1–BChE complex is at 429 nm, indicating that the porphyrin is in a much different environment (the active sites of the two proteins are different although each has a serine at the bottom). Unfortunately, the trough and the peaks may be of similar intensity [4], indicating that the oscillator strength (extinction coefficient) of the enzyme-bound porphyrin is similar to that of the porphyrin in water even though its absorbance spectrum is altered. Unfortunately, this does not increase the absorbance values.
Q-Bands
5.1. Absorbance by excited states: multiple photon absorbance Fig. 5. Energy level diagram of absorbance and emission dynamics of TPPS in water. Dashed downward lines represent non-radiative decay from singlet excited states; heavy upward lines represent absorbance of the singlet and triplet excited states.
1.5
ABSORBANCE
The absorbance of a photon in the B- or Soret band of the porphyrin (400–450 nm) increases the energy of a ‘‘ground state” electron of the porphyrin macrocycle resulting in the formation of a singlet excited state; the first singlet S1 excited state absorption coefficient for tetraphenyl porphyrin (H2TPP) is 2.35-fold greater than that of the Soret ‘‘ground” or unexcited state absorption coefficient [15]. Indeed, the absorbance of yet a third photon has been demonstrated in porphyrin [16] to generate excited singlet state Sn at an even higher energy level, resulting in an even larger apparent absorbance by a single porphyrin; three photons are absorbed in the Soret region with the second and third experiencing greater absorptivity (cf. Fig. 5). Indeed, these three photons could (in the case of H2TPP) show the same absorptivity as four photons (of ground state absorbance of photon). In porphyrins, the singlet excited state can undergo intersystem crossing (IC) to a triplet state which is also capable of absorbing two photons; the spectrum of the triplet excited state is similar to that of the ground and singlet excited states [17] although the absorption coefficient of the triplet state of tetraphenylporphyrin (H2TPP) is also reported 1.5 times that of the singlet ground state absorptivity [15]. The net result is that a single photon absorbance can result in at least three additional photons being absorbed by a single molecule [18]. Reindl and Penzkofer [19] reported triplet to singlet IC as well as singlet to triplet IC; crossing from the second excited triplet state T2 or higher state to the S1 or higher state returns the electron to a condition where it can again absorb a photon. The transfer from different states can proceed due to the number of radiationless transitions that dissipate the energy and allow for multiple photon absorbance by a single porphyrin molecule. As seen in Fig. 6, excitation of TPPS at 413 nm (the Soret or Bband) in the ground state results in the formation of excited states which can absorb a photon at around 416–418 nm and with comparable intensity to the ground state. The ratio of excited state absorptivity to ground state absorptivity is different for different porphyrin structures [15]; H2TPP has a value of 1.5 [15] while we observe H2TPPS to have a ratio of unity in the Soret band absorbance range. Gratz and Penzkofer [16] indicate that essentially the same fluorescence spectra are obtained from excitation at the B-band
Not Illuminated
Illuminated at 413 nm
415 nm peak
419 nm peak
1
.5
0 410
420
430
440
WAVELENGTH (nm) Fig. 6. Ground state (solid line) and excited singlet state (413 nm excitation) spectra (dashed line) of 3 lM TPPS in water. Spectra recorded with CARY 4E UV–vis spectrophotometer.
(405 nm) and the Qa(1) band (543 nm) of H2-tetraphenylporphyrin (H2TPP); in other words, excitation at the B-band decays to the Qband before intersystem crossing (IC) to the T1 triplet state; similar results were obtained by Karotki et al. [20]. Since the excited state of the Qa band is expected to be similar if achieved by direct excitation or decay from the B-band, it may be possible for excited Q to absorb a photon and enter a higher excited state (cf. Fig. 5). In the complete ‘‘cycle” from excitation of and removal of an electron from the ground state following absorbance of a ‘‘Soret” photon (all of our detection/sensor measurements utilize 413 nm light with a half-bandwidth of approximately 8–10 nm) until the electron ultimately returns to the ‘‘ground state”, at least five photons can be absorbed by the same single porphyrin. Thus, alteration of one porphyrin has the same absorbance intensity change as if five had been affected; this is essence an ‘‘amplification” of the detection event. A single porphyrin affected exhibits the same
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H.J. Harmon / Methods 46 (2008) 18–24
absorbance change of several porphyrins being affected; detection of 56 ppt or less is the same as for 280 ppt monomeric porphyrin absorbing one and only one photon assuming all excited states absorb as strongly as the ground state, which they do not. Factoring the increased absorptivity of the singlet and triplet states (as seen in H2TPP), a single porphyrin could absorb up to seven photons, the same as seven porphyrins each absorbing a single photon in the ground state; 40 ppt shows the same absorbance change as 280 ppt. 5.2. Upconversion is evidence of multiple photon absorbance Upconversion is a result of multiple photon absorbance when the excited state formed by absorbance of 2- (or 3) photons decays to ground state (or near ground state levels) by emitting a photon of greater energy (shorter wavelength) than either of the absorbed photons (the two photons absorbed need not be the same wavelength). Alternatively, the loss of energy may occur by a radiationless transition where no photon is emitted). The emission of a shorter wavelength photon is evidence that multiple photon absorbance has occurred. Extensively studied in Zn-porphyrins [21–23], the upconversion effect is also observed in non-metalloporphyrins [21,24–28]. Table 1 indicates the emitted upconverted photon wavelengths for each excitation wavelength of the Q- and near infra-red regions of H2TPPS at pH 8.6. The wavelengths in the 300–360 nm region are similar to the Nand L-UV absorbance bands of porphyrins. Similarly, the emissions at 642 and 704 nm correspond to an emitted photon as the electron moves from the T1 triplet excited state to the ground state. While we did not observe emission of a photon from 636 nm excitation of TPPS, multiple photon absorption resulting from absorbance of 700–1300 nm wavelength photons has been observed in other porphyrins [28]. This indicates that non-metallated porphyrin can also absorb multiple photons. Multiple photon absorption is typically performed with lasers at sufficient power to deliver a second photon during the lifetime of the excited state. The lifetime of the S1 singlet excited states in TPP is about 20 ns while the fluorescence lifetime is 9 ns; the lifetime if the T1 triplet excited state is 800 ns [15]. This allows continuous illumination at intensities far less that those of lasers to effect multiple photon absorbance. Furthermore, the absorbance crosssection (corresponds to the extinction coefficient) of the excited state is greater that of the ground state absorbance [15]; Drobizhev et al. [24] indicate weak ground state absorbance of the Q- and near IR bands but strong excited state absorbance at the same wavelengths (as the ground state). As a result, multiple photon absorbances can be observed using the continuous output light sources normally found in commercial spectrophotometers and fluorometers. However, multiple absorbance by excited states showing greater absorptivity does not explain the detection of the absorbance change corresponding to 7 ppt analyte [7] and thus 7 ppt porphyrin. Another mechanism may be likely involved.
Table 1 Emitted/fluoresced photons by TPPS as a result of multiple photon absorbance at various wavelengths Excitation wavelength (nm)
Emitted upconverted photon wavelength (nm)
517 554 582 636 700 750
305 321 355, 550 — 642 642, 704
5.3. Structure-induced absorbance changes The role of molecular structure in multiple photon absorbance should be considered. Changes in the symmetry or structure, such as a distortion, of a porphyrin can affect its absorbance. Two-photon absorbance in the Q-bands can be ‘‘forbidden”; the low extinction coefficient or absorbance cross-section is a result of a ‘‘forbidden (or non-favorable) transition”; this prohibition can be removed if the symmetry of the molecule (in this case, a porphyrin) increases either due to parity changes, spin interactions, or molecular structural distortions. In our research using monosulfonatotetraphenyl porphyrin (TPPS1) non-covalently bound into the active site of enzymes [2–12], small shifts in the absorbance maximum of the porphyrin occur upon binding to the enzyme. The shift is different for each different enzyme (cf. Table 2 below) since the microenvironment and the change in structure (distortion) of the porphyrin will be different in each enzyme active site; the wavelength maximum of non-bound TPPS1 is 405 nm. It is likely that the 7–21 nm red-shift of absorbance wavelength maxima reflects a change in the symmetry or structure of the porphyrin; this shift could increase the two-photon absorbance. Harmon [1] reported both the bathochromic (to longer wavelength) and hypsochromic (to shorter wavelengths) of tetraphenylsulfonate porphyrin (TPPS) on binding to different proteins. The phenyl rings on the porphyrin likely change their position and orientation to the porphyrin macrocycle plane, increasing the coupling of electronic and vibronic wavefunctions, contributing to an increase in multiphoton absorbance [28]. Karotki et al. [20] explain that a decrease in electronic symmetry can occur due to solvent effects (as would likely occur when the TPPS1 molecule leaves the active site and enters the aqueous medium) resulting in a change in two-photon absorbance cross-section. Similarly, a change in the dipole moment of the porphyrin as would occur as it associates with and dissociates from the enzyme will affect symmetry and the two-photon absorbance. Drobishev et al. [27] indicate drastic differences in two-photon absorbance in porphyrins with different electronaccepting groups at the periphery; this suggests that a change in electron distribution of the porphyrin will result in a change in two-photon absorbance intensity as well as alterations in the ground state spectrum. Thus, it is likely that changes in absorptivity in excited state absorbance may be the result of changes in porphyrin structure and electron distribution but will not on its own increase the sensitivity of detection beyond what has been presented thus far. 5.4. Concentration dependence of non-linearity of evanescent absorbance The detector response frequently is not linear with the concentration of the analyte being detected; instead, the change in absorbance may be proportional to the log of the analyte concentration (e.g., Refs. [3,4,10]). The apparent absorbance changes are significantly greater at lower concentrations than at higher concentrations. This is another ‘‘deviation” from the Beer–Lambert absorbance relationship; at higher concentrations of analyte, the absorbance change decreases. Such a deviation has been observed
Table 2 Wavelength maxima of TPPS1 incorporated into the active site of different enzymes Enzyme
Wavelength maximum of the enzyme–TPPS1 complex (nm)
AChE (acetylcholine esterase) BChE (butyrylcholine esterase) OPH (organophosphate hydrolase) CBPA (carboxypeptidase A)
426 421 412 423
H.J. Harmon / Methods 46 (2008) 18–24
by others [29–35] performing evanescent absorbance measurements using planar waveguides; the apparent evanescent absorbance exceeds bulk solution-based (e.g., with 1 cm cuvettes) absorbance of the same material. The basis of this has been addressed [29,36] with evanescent absorbance in coated optical fibers. Ruddy et al. [29] showed that the evanescent absorbance of a fixed analyte concentration varies linearly with the interaction length; they also indicated that at low analyte concentrations, the evanescent absorbance can be over 10 times greater than the bulk absorbance effect in 1 cm cuvettes. The net effect from these studies is that with low concentrations of analyte absorbed onto a planar waveguide, the evanescent absorbance can exceed bulk absorbance of the same material over 10-fold. This allows for accurate measurement of low analyte concentrations; the apparent measured absorbance for a given analyte concentration is greater than expected from the bulk Beer–Lambert relationship, as we have reported previously. 5.5. The role of self-quenching in evanescent thin film sensor response The enhancement of absorbance in evanescent measurement of thin films may also be due to the limitation of ‘‘quenching” reactions of the porphyrin. In solution, the porphyrin will absorb a photon in the ground state and enter an excited state; the excited state can decay via a radiationless transition within the porphyrin and/ or absorb a photon and move to a higher energy state (from the singlet or triplet state) or possibly become ‘‘self-quenched” where the energy of the excited state is transferred to another porphyrin molecule during a collision in solution. Thus, the absorbance in solution is limited by the number of collisions with adjacent molecules and may contribute to the non-linear deviation from Beer’s Law commonly observed (absorbance decreases at higher porphyrin concentrations); the deviation from linearity is also attributed to formation of dimers or aggregates. In a thin film of immobilized porphyrins or porphyrins surrounded by protein (as in the case of TPPS1 bound in the active site), the ability to freely collide with and transfer energy to adjacent porphyrins is severely limited. If intramolecular energy transfer cannot occur, the possibility for multiple photon absorbance to occur increases, enhancing the apparent absorbance intensity as explained earlier. We would expect that at higher density of porphyrins on the thin film, the possibility of energy transfer to an adjacent molecule will increase, limiting the photon absorbance to exciting a ground state electron only. In solution, porphyrins can interact and transfer energy in all directions of space while in the thin film interaction is limited to the plane of the surface (monolayer of absorber) and is facilitated by higher loading of the surface resulting in closer proximity of the energy acceptor molecules. The observed effect would be that the evanescent absorbance would increase with the amount of bound material until which time the excited molecules are able to freely interact with neighboring molecules, returning the donor molecule to the (single-photon-absorbing) ground state and exciting the acceptor into an excited state without benefit of a further (multiple) photon absorbance; the amount of light absorbed is thus less than if there were no interaction and the slope of absorbance vs. concentration curve would decrease with increasing analyte concentration, as observed [29]. Lakowicz and Weber [37] observed that I quenching of ethidium bromide fluorescence when bound to double helical DNA is 1/30 as effective as when the dye is in solution. They concluded that the quenching mechanism involves a quenching radius equivalent to the collisional radius regardless of the medium. Suh and Chaires [38] performed similar experiments and concluded that the fluorescent donor and acceptor (quencher) must be within a certain distance of each other for energy transfer to occur (this
23
can be a short distance in free solution but may be large in immobilized systems. Maniara et al. [39] studied phosphorescence of ANS (1-anilinonaphthalene-8-sulfonate) and TNS [(2-(p-toluidinyl)naphthalene-6-sulfonate] in solution and bound to bovine serum albumin (BSA) and, on the observation that phosphorescence is not observed in solution, concluded that molecules undergo rapid non-radiative decay from the singlet when free in solution. Since this competes with triplet formation, absorbance of photons in the singlet and triplet states will not occur; hence, enhanced absorbance will not be seen. Turro et al. [40] observed that the phosphorescence lifetimes of 1-bromonaphthalene and 1-chloronaphthalene increase when bound to cyclodextrin; the net result is that immobilization increases the lifetime of excited states, allowing a greater probability for photon absorbance by the excited state. Similarly, quenching of the sex steroid d1,3,5(10),6,8-estrapentaene-3,17b-diol [hydroequilenin (DHE)] by acrylamide decreased when the steroid was bound to rabbit sex steroid binding protein [41]. Vaughn and Weber [42] observed that the fluorescence of pyrenebutyric acid bound to BSA, hemoglobin, and polylysine is not affected by the concentration of oxygen, unlike when the fluor is in free solution. The rate of quenching of the excited state of the fluorescent molecules by collision with a quencher molecule is directly proportional to D, the summed diffusion coefficients of the fluor and quencher, N0 the number of molecules per volume, and a, the encounter distance (sum of molecular radii). The rate of quenching is expressed by the following expression:
k ¼ cð4paDN0 Þ
ð3Þ
with the factor of 4p indicative of the 3-dimensional aspect of the quencher affecting the fluorophore where c is the quenching efficiency. However, if one or both of the molecules are immobilized, the diffusion coefficient is drastically decreased. Thus the multiple photon absorbance and enhanced evanescent absorbance are related to each other at low sensor indicator (e.g., the porphyrin) concentrations where the effective distance between fluorescent donor and acceptor exceeds the effective transfer distance and where immobilization of the molecules restricts the ability to encounter each other and transfer energy. 6. Conclusion This report describes the selectivity and sensitivity of chemical and biological detectors by the use of a colorimetric indicator (porphyrin) which is incorporated into the active site of specific enzymes and incorporated into the biomimetic or naturallyoccurring binding/receptor site for the biologicals. In each case, binding of the porphyrin or its complex changes the electron distribution of the porphyrin which alters the absorbance spectrum of the porphyrin. The change in absorbance intensity is frequently non-linearly proportional to the amount of agent/analyte present and bound. On the basis of known and published extinction coefficients of the porphyrin, the limit of detection derived from Beer’s Law is approximately 2 nM although analytes can be detected at far lower levels. This apparent discrepancy between the expected and observed values is most likely the result of multiple photon absorbance by the porphyrin in the ground state and in multiple singlet and excited states such that a single porphyrin molecule can absorb multiple photons during the ‘‘cycle” of photon absorbance in the grounds state until the electron returns to or near the ground state levels. The outcome is ‘‘amplification” of the binding such that binding of a single analyte molecule to a single porphyrin results in the absorbance of several photons; the absorbance intensity mimics the effect of several porphyrins each absorbing a photon.
24
H.J. Harmon / Methods 46 (2008) 18–24
This is a deviation from the Beer–Lambert relationship. The more typical deviation form the relationship is observed when porphyrins aggregate; the apparent extinction coefficient decreases with increasing porphyrin concentration as a result of aggregation, each aggregate absorbing light as if it were a single porphyrin. In the present instance, the deviation from the Beer–Lambert relationship is opposite: a single porphyrin absorbs light as if it were multiple single-photon-absorbing porphyrins; this is a type of non-linear optics. This optical absorbance amplification is consistent with the non-linear optical properties of porphyrins and related molecules. The use of multiple photon absorbance can significantly increase the sensitivity of optical absorbance-based detectors. The absorbance of multiple photons coupled with the enhanced absorbance in evanescent measurements because of minimization of intramolecular energy transfer and energy quenching combine to give increased apparent measurable absorbance in our evanescent sensors. The combined effect is expected to be multiplicative; thus measurements of concentrations 10- to 100-fold less than expected in bulk solution measurements (using the same extinction/attenuation coefficients) can be expected depending on the number of photons absorbed by the porphyrin. Such an expectation is consistent with the observed results where the observed absorbance change is much greater than that predicted from simple Beer–Lambert type absorbance-concentration relationships. The nature of enzyme-based sensors has been reviewed recently [9,12]. The function of these sensors is a wondrous combination of chemical interactions, biochemical enzymatic specificities, and non-linear optical absorbance. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
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