ISCEV guidelines for clinical multifocal electroretinography (2007 edition)

The clinical multifocal electroretinogram (mfERG) is an electrophysiological test of local retinal function. With this technique, many local ERG responses, typically 61 or 103, are recorded from the cone-driven retina under light-adapted conditions. This document specifies guidelines for performance of the test. It also provides detailed guidance on technical and practical issues, as well as on reporting test results. The main objective of the guidelines is to promote consistent quality of mfERG testing and reporting within and among centers. These 2007 guidelines, from the International Society for Clinical Electrophysiology of Vision (ISCEV: http://www.iscev.org), replace the ISCEV guidelines for the mfERG published in 2003.
Introduction
The electroretinogram (ERG) is a mass potential, the result of the summed electrical activity of the cells of the retina. Full-field electroretinography is a well-established clinical technique for evaluating global retinal function [1]. The multifocal ERG (mfERG) technique was developed to provide a topographic measure of retinal electrophysiological activity. With this technique, many local ERG responses, typically 61 or 103, are recorded from the cone-driven retina under light-adapted conditions. In 2003, the International Society for Clinical Electrophysiology of Vision (ISCEV) published guidelines for recording the mfERG [2]. These were guidelines, not standards, to allow for further research before standards were set.
Although the mfERG now has been used for more than 10 years to aid in the diagnosis of diseases of the retina, ISCEV decided that it is still premature to set specific standards. However, to take into consideration recent developments in technology and practice, this document provides revised guidelines for recording clinical mfERGs. These guidelines will be reviewed periodically, consistent with ISCEV’s practice.
Description of multifocal electroretinography
The mfERG technique is a method of recording local electrophysiologic responses from different regions of the retina. Electrical responses from the eye are recorded with a corneal electrode as in conventional, full-field ERG recording. However, the nature of the stimulus and the form of the analysis differ. These differences allow a topographic map of local ERG activity to be measured. For the basic mfERG described here, the retina is stimulated with an array of hexagonal elements, each of which has a 50% chance of being illuminated every time the frame changes (Fig. 1). Although the pattern appears to flicker randomly, each element follows the same pseudo-random sequence of illumination with the starting point displaced in time relative to other elements. By correlating the continuous ERG signal with the sequence of on- and off-phases of each element, the local ERG signal is calculated. Although we will refer to these local ERG signals as mfERG responses, it is important to keep in mind that they are not direct electrical potentials from local regions of retina, but rather they are a mathematical extraction of the signal. Further, because the stimulation rate is rapid, the waveform of the local mfERG response can be influenced both by preceding (‘adaptation effects’) and subsequent stimuli (‘induced effects’), as well as by the responses to light scattered on other retinal areas.
Fig. 1(a) Representative hexagonal mfERG stimulus array with 61 elements scaled with eccentricity. Roughly half of the elements are illuminated at any one time. (b) Same as in panel A for an array with 103 elements
The typical waveform of the basic mfERG response (also called the first-order response or first-order kernel) is a biphasic wave with an initial negative deflection followed by a positive peak (Fig. 2). There is usually a second negative deflection after the positive peak. These three peaks are called N1, P1 and N2, respectively. There is evidence that N1 includes contributions from the same cells that contribute to the a-wave of the full-field cone ERG, and that P1 includes contributions from the cells contributing to the cone b-wave and oscillatory potentials. Although there is some homology between the mfERG waveform and the conventional ERG, the stimulation rates are higher for the mfERG and, as noted above, the mfERG responses are mathematical extractions. Thus, the mfERG responses are not technically “little ERG responses”. Therefore, the designations ‘a-wave’ and ‘b-wave’, used for the full-field ERG, are not appropriate to describe features of the mfERG waveform.
Fig. 2Diagram of an mfERG response to show the designation of the major features of the waveform
Basic technology
Electrodes
Recording electrodes
Poor or unstable electrode contact is a major cause of poor quality records. It is important to follow the recommendations concerning fiber, foil, loop and contact lens electrodes in the full-field ERG Standard [1] and Pattern ERG (PERG) Standard [3]. In particular, electrodes that contact the cornea, or nearby bulbar conjunctiva, are required. In addition, good retinal image quality and proper refraction is desirable.
Reference and ground electrodes
Proper application of suitably conductive electrodes is essential for reliable mfERG recordings. Follow the recommendations made in other ISCEV Standards [1, 3].
Electrode characteristics, stability and cleaning
Follow the recommendations in the ISCEV full-field ERG and/or PERG Standards [1, 3].
Stimulation
Stimulus source
Until recently, the mfERG stimuli were most commonly displayed on a cathode ray tube (CRT), i.e. a monitor. CRT monitors are being rapidly replaced with other devices such as liquid crystal display (LCD) projectors, arrays of light emitting diodes (LEDs) and organic LEDs (OLEDs). These alternative modes of stimulation can affect the amplitude and waveform of the mfERG making it essential to specify the type of display when reporting results.1
Frame frequency
A CRT frame frequency of 75 Hz has been used widely. (LCD displays use 60 Hz.) Use of different frequencies can substantially alter the amplitude and waveform of the mfERG response. Whatever frame frequency is used, normative values for normal healthy subjects need to be determined separately for that frequency. Further, it is essential to specify the frame frequency when reporting results.
Luminance and contrast
In general, the luminance of the stimulus elements for CRT displays should be 100–200 cd/m2 in the lighted state and low enough in the dark state to achieve a contrast of ≥90%. This means that the mean screen luminance during testing will be 50–100 cd/m2. Although higher luminance levels can be used, the ability to detect local defects may be decreased due to the effects of stray light. In addition, the luminance requirements may differ if a non-CRT display is used. In any case, the surround region of the display (the area beyond the stimulus hexagons) should have a luminance equal to the mean luminance of the stimulus array.
Calibration
As with other electrophysiologic signals, luminance and contrast affect the recorded signal and it is important to calibrate the stimulus following ISCEV guidelines [4]. The luminance of the dark and the light stimulus elements should be measured with an appropriate calibrator or spot meter. Many monitor screens are not of uniform brightness over the entire screen. While some variation is to be expected, a variation of greater than 15% is considered unacceptable. Some commercial systems are equipped to calibrate the display. If this ability is not present, we urge manufacturers to provide instructions for calibration of their devices.
Stimulus parameters
Stimulus pattern: The typical mfERG display is a hexagonal stimulus pattern scaled in size to produce mfERG responses of approximately equal amplitude across the retina. Thus, the central hexagons are smaller than the more peripheral ones. Different patterns may be useful in special cases (e.g. equal size hexagons for patients with eccentric fixation). These guidelines cover only the typical stimulus pattern, scaled to produce approximately equal size responses for healthy control subjects.
Flicker sequence: Most commercial mfERG instruments use an m-sequence to control the temporal sequence (between light and dark) of the stimulus elements. An m-sequence, in which the elements can change with every frame, is recommended for routine testing. Different sequences, or the inclusion of global light or dark frames, have been suggested for specialized applications, but they are not a part of these guidelines, and should not be done in exclusion of a “standard” mfERG for routine clinical purposes.
Stimulus size and number of elements: The overall stimulus pattern should subtend a visual angle of 20–30° on either side of the fixation point. The stimulus region can be divided into different numbers of hexagons. The most frequently used patterns have 61 or 103 hexagons, with 241 hexagons occasionally used. The choice depends on balancing the need for good spatial resolution and a high signal-to-noise ratio, while minimizing the recording time. (See discussion below under clinical protocol).
Fixation targets: Stable fixation is essential for obtaining reliable mfERG recordings. Central fixation dots, crosses and circles are available with most commercial systems. The fixation targets should cover as little of the central stimulus element as possible to avoid diminishing the response. However, the examiner should always verify that the patient can see the fixation target. When the fixation targets are enlarged for low-vision patients, care should be taken not to obscure regions of interest. For example, a larger central cross will lead to a smaller central response simply due to occlusion of more of the stimulus.
Recording, analyzing and presenting results
Amplifiers and filters
The gain of the amplifier should produce recognizable signals without saturation. Appropriate band-pass filtering removes extraneous electrical noise, without distorting waveforms of interest. For a “standard” mfERG, the high pass cutoff can range between 3 and 10 Hz and the low pass cutoff between 100 and 300 Hz. Filter settings, even within the ranges suggested, will influence the response waveform. Thus, the filter settings should be the same for all subjects tested by a given laboratory, as well as for the norms to which they are compared. Line-frequency or notch filters should be avoided.
Signal analysis
Artifact rejection: Because blinks and other movements can distort the recorded waveforms, commercial software usually includes ‘artifact rejection’ algorithms to eliminate some of these distortions. When applying an artifact rejection procedure after the recording, care should be exercised to assure that clinically important aspects of the waveform are not being modified. In any case, artifact rejection procedures should be specified when reporting mfERG results.
Spatial averaging: In order to reduce noise and smooth the waveforms, some commercial programs allow the averaging of the response from each stimulus element with a percentage of the signal from the neighboring elements. Spatial averaging can help visualize the mfERG signal in noisy records. However, spatial averaging may obscure small, local changes or the borders of regions of dysfunction. Thus, it should be used with care and specified when reporting results. Further, the default conditions of commercial software should be examined, as spatial averaging may be a default condition.
Displaying results
Trace arrays: All commercial programs can produce an array of the mfERG traces (Fig. 3a, b). This display of the results is useful for visualizing areas of abnormality and for comparing the mfERG results to visual fields from perimetry. The trace array is the basic mfERG display and should always be included in the report of clinical results. Commercial software typically allows for these responses to be displayed with either a visual field view or retinal view as the frame of reference. The frame of reference should be indicated. Note that in Fig. 3, each of the 61 (panel a) or 103 (panel b) responses are shown in approximately correct topographic representation. Adding contours to show the loci of different eccentricities, as in Fig. 4, can help in determining whether the regions of low amplitude correspond to regions of the visual field that show a loss of sensitivity.
Fig. 3Sample mfERG trace arrays (field view) with 61 elements (panel a) and 103 elements (panel b). (c, d) The 3-D response density plots (field view) associated with panels a and bFig. 4The mfERG trace array (left panel, field view) and the probability plot from standard automated perimetry (right panel) for a patient with retinitis pigmentosa. The contours for a radius of 5 and 15° are shown. The light gray, dark gray, and black squares indicate statistically significant field loss at the 5, 1 and 0.5 percent levels, respectively
Group averages: Commercial programs allow for the averaging of groups of responses from the trace arrays. This can be helpful for comparing quadrants, hemiretinal areas, normal and abnormal regions of two eyes, or successive rings from center to periphery. The latter is particularly useful for patients who have diseases that produce approximately radially symmetric dysfunction. In addition, responses from stimulus elements associated with a local area of interest can be averaged for comparison with a similar area in an unaffected eye or to data from control subjects. Figure 5 shows the result of grouping the responses in Fig. 3b by rings. The most commonly used display is response density (Fig. 5a), in which the responses from the elements in each ring are summed and then divided by the area of these elements. Figure 5b and c are alternative representations of the data in which summed responses from each ring are shown (panel c) or are normalized so that they have the same amplitude (panel b).
Fig. 5The mfERG responses in Fig. 3b were grouped by concentric rings and summed to yield the ‘Summed’ responses in panel c. These summed responses are divided by the area of the elements of the ring for the ‘Response Density’ responses (panel a) and normalized so each has the same amplitude for the ‘Normalized’ responses (panel b)
Topographic (3-D) response density plots: The 3-D plot (Fig. 3c, d) shows the overall signal strength per unit area of retina. This display can sometimes be useful for illustrating certain types of pathology. In addition, the quality of fixation can be assessed by observing the location and depth of the blind spot. However, there are major dangers in using the 3-D plot in assessing retinal damage. First, information about the waveforms is lost. Thus large, but abnormal, or delayed responses can produce normal 3-D plots. Second, a central peak in the 3-D plot can be seen in some records without any retinal signal (see, Appendix: Artifact recognition examples for electrical noise and weak signals). Finally, the appearance of the 3-D plot from a given recording is dependent on how the local amplitude is measured. Whenever 3-D plots are presented, the method used to measure local amplitude should be identified, and the corresponding mfERG trace array also should be presented.
Signal extraction: Kernels—This document is aimed at the general mfERG user and only describes the basic response, the first-order kernel. Higher order kernels, particularly the second-order kernel, occasionally are reported, and used in special applications.
Clinical protocol
Patient preparation
Pupils
The pupils should be fully dilated and pupil size noted.
Patient positioning
Subjects should sit comfortably in front of the screen. Relaxation of facial and neck muscles will reduce artifacts from muscles; a headrest may be helpful. The appropriate viewing distance will vary with screen size, in order to control the area (visual angle) of retina being stimulated.
Fixation monitoring
Good fixation, both central and steady, is essential. Thus, fixation should be monitored, preferably by the use of monitoring instrumentation available on some units. When this option is not available, careful direct observation may be employed.
Refraction
Although there is some evidence that the mfERG is unaffected by moderate blurring of the retinal image in healthy individuals, we recommend refraction for optimal acuity. On some commercial machines, a manual adjustment of the viewing optics is possible. Alternatively, lenses can be placed in a holder positioned in front of the eye. In the latter case, the viewing distance must be adjusted to compensate for the relative magnification of the stimulus. Also care must be taken to avoid blocking the view of the stimulus screen by the rim of the lens or the lens holder and thus creating an apparent scotoma.
Monocular versus binocular recording
Recording is typically done with monocular stimulation. Those who record binocularly should be aware that signals can be altered by misalignment of the eyes.
Adaptation
Pre-adaptation (before test): Subjects should be exposed to ordinary room lighting for at least 15 min prior to testing. Longer adaptation times may be needed after exposure to bright sun or bright lights such as those used for fundus photography or retinoscopy.
Room illumination: Moderate or dim room lights should be on and ideally should produce illumination close to that of the stimulus screen.
Stimulus and recording parameters
Stimulus Size
The stimulus should subtend 20–30° of visual angle on either side of the fixation point.
Number of elements
A display containing 61 or 103 elements should be used.
Duration of recording
A total recording time of at least 4 min for 61 element arrays, or 8 min for 103 element arrays, is recommended, although these times might be adjusted by experienced laboratories according to clinical needs. The overall recording time is divided into shorter segments (e.g. 15–30 s) so that subjects can rest between runs if necessary and also so that a poor record (from noise, movement or other artifacts) can be discarded and repeated without losing prior data.
Trade-offs
Various manipulations will affect the signal-to-noise ratio (SNR) of the responses. In particular, decreasing the size (increasing the number for a fixed stimulus field size) of the stimulus elements and decreasing the duration of the recording will decrease the SNR of the responses. While decreasing the number (increasing the size) of elements will increase the SNR, it will decrease the spatial resolution of the test. In general, conditions with larger (i.e. 61) elements and a shorter recording time (e.g. 4 min) are easier for the patient and suitable for a general screening of macular function. On the other hand, conditions with 103 elements and a longer recording time (e.g. 8 min) are useful for assessing foveal function and mapping the outline of retinal defects. Very small elements (such as a 241 hexagon array) may sometimes be helpful for diseases with very small or irregular effects. Repeat recording is recommended to confirm small or subtle abnormalities.
Further, the choice of electrode type will also influence the SNR of the responses. For example, bipolar corneal contact electrodes yield recordings with the highest SNR. Thus, longer recording times, repeat measurements and/or fewer stimulus elements are necessary to obtain comparable SNRs when a foil or fiber electrode is used.
Data reporting
Mode of display
Trace arrays: It is essential to show the trace array when reporting mfERG results (see Fig. 3a, b). These arrays not only show topographic variations, but also demonstrate the quality of the records, which is important in judging the validity of any suspected variations from normal. Trace lengths of 100 ms or more should be used for these displays. (It is hard to detect interference from line frequency and/or kernel overlap in shorter trace lengths.).
Group averages: Arranging responses by groups can be a useful way to summarize the data. Concentric rings of traces, from the center outward, are most commonly used. Regions with fundus pathology can be averaged together if desired. Most laboratories report response density (Fig. 5a).
Three-dimensional plots: These should be used with caution and only when accompanied by trace arrays (see above). The 3-D plots (Fig. 3c, d), without accompanying trace arrays, can be misleading (see Appendix). Note that if fixation is steady and central, a clear depression due to the blindspot should be present and located in the appropriate place.
Measurements calibration marks: Calibration marks must accompany all traces or graphs. This will enable comparisons among patients or within a patient on sequential visits.
Measuring mfERG amplitude and timing: The N1 response amplitude is measured from the starting baseline to the base of the N1 trough; the P1 response amplitude is measured from the N1 trough to the P1 peak (see Fig. 2). The peak times (implicit times) of N1 and P1 are measured from the stimulus onset. Measurements of group averages should routinely include the N1 and P1 amplitudes and peak times.
Commercial software provides measures of the overall amplitude and timing of the mfERG traces. There are various procedures for measuring amplitude (e.g. trough-to-peak amplitude), latency (e.g. response shifting, response stretching, time to peak), or overall response waveform (e.g. scalar product, root-mean-square (RMS)). A description of these techniques is beyond the scope of these guidelines. However, it should be noted that when a template is needed (e.g. for scalar product measures), the template should be formed from age-similar control data obtained from that laboratory.
Color scales: The use of color scales is optional; care should be taken when reproducing color images on a gray scale as the luminance sequences may not be in the proper order.
Normal values
Each laboratory must develop its own normative data. Variations in recording equipment and parameters make the use of data from other sources inappropriate. Because electrophysiologic data are not necessarily described by a normal distribution, laboratories should report the median value rather than the mean, and determine boundaries of normality. The mfERG, like the full-field ERG, is somewhat smaller in amplitude in older individuals and in those with highly myopic eyes. Although these effects are generally not large, they can be important in the evaluation of some patients. In any case, age-adjusted normative data is recommended.
Reporting of artifacts and their resolution
Reports should indicate any problems with the recording such as movement, head tilt, poor refraction capability, poor fixation, etc. that might affect reliability and interpretation. Also, indicate explicitly any artifact reduction procedures or post-processing maneuvers used to prepare the data. This should include the type and number of artifact rejection steps, the spatial averaging with neighbors (noting the extent and number of iterations), and any other averaging or filtering procedures.

multifocal electroretinogram
electroretinogram
clinical guidelines


Activation kinetics of single P2X receptors

After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X2, P2X4, and P2X7 receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level.
Primarily, P2X receptors are ligand-gated ion channels. This ion channel family, also known as ion channel-coupled receptors, mediates the fastest known signal transduction from extracellular messengers to the intracellular environment. This signal transduction mechanism is so simple (i.e., includes few steps) that it becomes fully activated in the low millisecond range. The best known biological structures using this approach are neuronal synapses in which receptors for the classic transmitters acetylcholine, glutamate, gamma-aminobutyric acid (GABA), glycine, or serotonin transmit excitation from one neuron to another in less than 1 ms [1]. This rapid activation kinetics of the ligand-gated ion channels is too fast to become analyzed by whole-cell current recordings or even slower fluorescent dye-based measurements of changes of the intracellular ion composition. The main reason for the low speed of these recording techniques compared to the receptor kinetics is the rate-limiting solution exchange at the extracellular side of the cell membrane where the ligand binding site is located. These obstacles in the investigation of ligand-gated channels can be overcome by the single-channel patch clamp technique where tiny parts (patches) are excised out of the cell membrane containing at best only one receptor. In such preparations, solution exchanges can be achieved within less than 1 ms [2]. Apart from the high time resolution, this technique has a resolution of ionic currents in the pA (picoampere) range sufficient to observe the ionic flow through and the gating (opening and closing) of single ion channels. With this technique, ion channels can be characterized and identified according to their conductance, macroscopic kinetics (time course of activation, i.e., opening of channels after ATP application, and of deactivation, i.e., closing of channels after ATP withdrawal), and microscopic kinetics (i.e., the characteristic mean times channels spend in closed or open configurations). The ultimate goal of the kinetic analysis is the development of a kinetic (normally Markovian) model of closed and open states which describes the agonist concentration dependency of the macroscopic and microscopic kinetics. Additionally, single-channel analysis may be able to discriminate whether various effects of agonists and antagonists are caused by changing agonist binding or efficacy or by altering the ion channel permeation behavior [3].
Additionally, patch clamping allows the separation of currents flowing through the ligand-gated channels from contaminating currents mediated by conductances activated downstream by secondary signals. In the case of P2X receptors, such signals may be changes in the intracellular concentration of Ca2+, Na+, K+, or H+ ions which can permeate the channel pore. The next steps in signal cascades may be changes of the cell volume with effects on the cytoskeleton, oxidative stress, and furthermore activation of kinases, phosphatases, or transcription. Therefore, investigating the time course of opening and closing of P2X receptors may help to understand signaling cascades they activate. It may also detect functional changes induced by receptor mutations more precisely than whole-cell current recordings or measurements of P2X receptor-induced changes of the intracellular Ca2+ concentration or even later effects.
Soon after the introduction of the patch clamp technique, ATP-activated single channel events were discovered and analyzed. They were first described in rat sensory neurons [4] and cultured chicken myoblasts [5]. Later, different ATP-activated single ion channel currents were reported in smooth and cardiac muscle, glands, and other neurons where single-channel conductances between <1 and 60 pS were measured [6].
More detailed information about P2X receptor kinetics was obtained from measurements of the rise time of ATP-induced whole-cell currents which, as typical for ligand-gated receptors, decreased with increasing agonist concentration in the range of 5–25 ms [7, 8]. A more sophisticated kinetic analysis of P2X receptor function was performed in bullfrog dorsal root ganglion cells. The concentration dependence of the activation and deactivation time course as well as of the steady-state current during prolonged ATP application could well be described by a linear Markov model where the independent binding of three ATP molecules to equal binding sites leads to the opening of the channel which in the triliganded state is open 25% of the time:
Here, the rate constants are given in s−1 and β/α = 1/4 [9]. Remarkably, the trimeric structure of cloned P2X receptor channels described much later [10–12] implies the necessity of binding of three ATP molecules and therefore coincides well with this model.
Investigations of the microscopic kinetics of native P2X receptors were used to characterize aspects of the gating behavior of the channels in distinct preparations but were not developed into kinetic models [5, 13–18].
After cloning the P2X receptor subtypes, the kinetic analysis of heterologously expressed distinct P2X receptors became principally possible. However, the collection of enough single-channel data for establishing a kinetic model turned out to be too laborious or even seemed to be impossible on receptors which desensitize quickly (within milliseconds) and recover slowly (within minutes) [19, 20]. Therefore, models for P2X1 [21] and P2X3 [22] receptors have been developed which, like the model developed by Bean [9], are based on whole-cell recording and include additional desensitized receptor states.
For the slowly desensitizing P2X4 receptor, the effect of external Mg2+ ions was investigated on the single-channel level. The blocking effect of Mg2+ could be partially ascribed to the reduction of the mean open time [23]. A more detailed analysis of the single-channel kinetics of P2X4 receptors revealed three different mean open times and five mean closed times following activation of the receptor by 0.3 μM ATP, where desensitization is slow. Hence, it was concluded that a kinetic model should have at least three open and five closed states [24].
A more comprehensive investigation of the single-channel kinetics under steady-state conditions (i.e., during prolonged application of constant extracellular ATP concentrations where the distribution of conformational states of receptors is not changing with time) was performed on the very slowly desensitizing P2X2 receptor [25]. It should, however, be mentioned that P2X2 receptors may exhibit substantial fast desensitization in excised patches [26]. The following model was developed:
In contrast to Bean’s model [9] the three ATP binding sites are not equal in this case. Specifically, the second binding step possesses a higher association rate constant and a much higher dissociation rate constant compared to the first. Both together result in a decreased affinity. This could be interpreted in such a way that the binding of ATP at the first site induces a conformation change that leads to an easier accessibility of a second binding to ATP. But this second binding is weaker, possibly due to the repulsion of both anionic ATP4− molecules. Negative cooperativity due to ATP repulsion was also suggested for the human P2X7 receptor [27]. The addition of an open (O6) and a closed state (C7) to a simple C–C–C–C–O model was necessary to describe openings of the P2X2 receptor which occurred as single openings or as bursts. This model was however unable to explain P2X4 receptor kinetics of multichannel patches, and therefore a positive cooperativity toward opening of P2X4 receptor channels was assumed [28].
The P2X7 is another non-desensitizing P2X receptor which proved to be suitable for kinetic analysis [29]. A relatively simple model was used to describe both the ATP dependence of microscopic (open and shut times, open probability of single channels) and macroscopic kinetics (activation and deactivation time course) of the main gating mode measured by a combination of single-channel recording and an ultrafast solution exchange system:
The model could be simplified compared to that describing the kinetics of the P2X2 receptor, since the open time distribution could be described in most cases by one exponential component and the channel did not display a bursting behavior. The much lower association rate constants compared to the P2X2 model are owed to the low ATP affinity of the P2X7 receptor. The kinetics of a second gating mode of single P2X7 receptors were about four times slower but could principally also be described by this model.
The assumption of only two ATP binding sites was sufficient for the approximation of the measured P2X7 receptor kinetics. This is a further deviation from the models mentioned above. The finding that P2X7 receptors are putative trimers like other P2X receptors and therefore contain probably three ATP binding sites seems to be contradictory to the P2X7 receptor kinetics model. As yet, a comprehensive explanation cannot be given but there are indications for a third binding site that induces very small single ion channel currents (Riedel et al., unpublished).
Mean currents calculated by this model displayed an approximately monoexponential activation and deactivation time course with time constants in the range of 10–25 ms. It was much simpler than the kinetics of several measured P2X7 receptor-dependent whole-cell currents [11]. This indicates that under whole-cell conditions either other ion currents may become secondarily activated or the biophysical properties of single P2X7 receptors themselves become less complex due to washout of an essential receptor component. However, even in cell-attached recordings, in which the intracellular environment is preserved, the single-channel current measurements at P2X7 receptors gave indication neither of more complex activation kinetics nor of a dilatation of the channel pore which is assumed to occur during prolonged ATP application [11]. Moreover, these single-channel current characteristics are indistinguishable from those measured in human lymphocytes [16], corroborating the view that the native human P2Z receptor is a genuine P2X7 receptor. Detailed analysis of the permeation characteristics [30] revealed a stable single-channel conductance even in external Na+-free and low Ca2+ solutions during long-lasting application of large ATP concentrations where a permeability of the cell membrane to large organic cations was induced in whole-cell preparations [11, 31, 32]. Instead, the permeation behavior and the calculated pore size of about 8.5 Å was similar to P2X1 and P2X2 receptors [33, 34]. Accordingly, it can be concluded that the apparent pore dilatation of P2X7 receptors observed in macroscopic current recordings [32] has no equivalent at the single-channel level, suggesting that the P2X7 receptor-induced permeability increase is not due to pore dilatation but reflects the induction of additional conductances mediated by other proteins secondary to P2X7 receptor activation. As recently reported, such additional conductance may be established by pannexin [35, 36], which can be activated by intracellular Ca2+ ions [37] and membrane stretch [38]. Similarly, single-channel recordings from P2X2 [33] and P2X4 receptors [24] do not indicate any pore dilatation which is assumed to occur in whole-cell preparation [39–41].
Instead of changing the permeation behavior, extracellular Na+ removal was found to drastically change the P2X7 ion channel kinetics [30]. The open probability, the mean open time as well as the activation and deactivation time constants considerably increased when Na+ was replaced by other monovalent cations. This effect was highly specific to Na+, suggesting that Na+ is not only a permeating ion but also a modulator of P2X7 receptor gating. The fact that the P2X7 receptor-induced increase of the cell membrane permeability to organic cations is promoted in Na+-free media [42, 43] can therefore be explained by an enhanced P2X7 receptor activation. This results in an increased Na+ and Ca2+ influx that may activate further permeation pathways.
It turned out that the kinetic model for P2X7 receptors [29] could easily explain the potentiating effect of Na+ removal, assuming that binding of Na+ to an extracellular site reduces the rate constant from O4 to C3 from 200 s−1 to 8 s−1. Although this is an allosteric effect not interfering with ATP binding, the concentration-response curve is shifted leftwards by Na+ removal, giving rise to a reduced apparent ATP binding constant. Such an apparent increase of a binding site affinity due to an increased efficacy has already been described by Colquhoun [44]. In this respect, single-channel recording has again proven to be the (possibly only) method to quantitatively measure and distinguish effects on single proteins of drug binding and the following conformational changes considered as efficacy. The model explains on the molecular level the known stimulating effect of Na+ substitution on effects mediated by P2X7 or P2X7-like native receptors [11].
Under conditions of hypoxia or tissue injury, ATP and K+ are secreted into the extracellular space [11]. Therefore, in metabolically compromised tissues, a replacement of extracellular Na+ by K+ may enhance the efficacy and potency of ATP on P2X7 receptors. Furthermore, ATP-mediated cell depolarization increases the driving force for K+ efflux and lowers the EC50 value of Na+ since the site seems to be located in the electrical field of the membrane [30]. In this way, K+ efflux may reinforce the ATP effect on P2X7 receptor-expressing cells, and even low concentrations of ATP may activate the P2X7 receptor to a substantial extent. However, whether such a mechanism is of relevance in vivo remains to be established.
In the future, the combination of single-channel recordings and molecular engineering methods will reveal more details concerning the function of P2X receptor-dependent ion channels. To this end, it may help to dissect and understand early and late steps of the signaling cascades which are started with the binding of ATP and are followed by biological processes known to be influenced by P2X receptors like contraction, secretion, perception, proliferation, and apoptosis [45]. Furthermore, patch clamp recording will continue to describe the effects of P2X receptor modulators on the single-channel level (for review of the investigations already performed, see North [11]).

kinetics
p2x4
p2x7
model
single channel
purinergic receptor
voltage clamp


Control of excitatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice

Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 μM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 μM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice.
1
Introduction
Capsaicin, the pungent ingredient of red peppers, severely affects pain sensation, inflammation or hyperalgesia. Systematic studies aiming to elucidate the effects of capsaicin revealed that this alkaloid primarily targets sensory fibers of the C type, where it activates a member of the transient receptor potential (TRP) channels, TRPV1 vanilloid receptors (TRPV1Rs) (Szallasi and Blumberg, 1999; Szolcsanyi, 2004). These receptors, cloned by Caterina et al. (1997), are non-selective cation channels gated by heat, low pH or endogenous ligands, such as anandamide (Tominaga et al., 1998; Zygmunt et al., 1999; Caterina et al., 1999; Smart et al., 2000). In addition, capsaicin could also affect the operation of both voltage-gated sodium and calcium channels (Balla et al., 2001; Lundbaek et al., 2005; Kofalvi et al., 2006), indicating that some of the capsaicin effects might not be linked to TRPV1Rs.
In contrast to the well-established function of TRPV1Rs in the periphery, its role is much less obvious in the central nervous system. Using autoradiography or immunohistochemistry, TRPV1Rs were shown to be present in several brain regions, including cortical structures (Acs et al., 1996; Mezey et al., 2000; Roberts et al., 2004; Toth et al., 2005; Cristino et al., 2006). Importantly, the specificity of signals in two of these reports has been confirmed in TRPV1R knockout mice (Roberts et al., 2004; Cristino et al., 2006), strongly arguing for the existence of TRPV1Rs in the CNS, yet their subcellular (synaptic or extrasynaptic) localization remains to be determined by high resolution electron microscopy. The functional role of TRPV1Rs in distinct brain regions was addressed by electrophysiological experiments. In the hippocampus, for example, an increase in paired-pulse depression of field potentials after application of capsaicin or anandamide has been noticed, and this effect was found to be sensitive to TRPV1R antagonists (Al-Hayani et al., 2001; Huang et al., 2002). In addition to the investigation of capsaicin effects on field potentials, other studies have examined the activation of putative TRPV1Rs on synaptic transmission more directly. These studies have found that in distinct parts of the brain, glutamatergic, but not GABAegic synaptic communication could be controlled by capsaicin, an effect that was also antagonized by TRPV1R antagonists (Sasamura et al., 1998; Hájos and Freund, 2002; Marinelli et al., 2002, 2003; Xing and Li, 2007). Interestingly, excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation were found to be depressed after application of capsaicin, whereas the same treatment significantly increased the occurrence of spontaneous EPSCs without affecting their amplitude.
To reveal whether the effect of capsaicin on synaptic glutamate release is indeed mediated by TRPV1Rs, we investigated the properties of EPSCs in dentate granule cells after bath application of this prototypic vanilloid ligand both in wild type and TRPV1R knockout mice.
2
Experimental procedures
Experiments were carried out according to the guidelines of the institutional ethical code and the Hungarian Act of Animal Care and Experimentation (1998. XXVIII. section 243/1998). Wild type and TRPV1R knockout mice of both sexes (20–77 days old, C57BL/6J strain) (Davis et al., 2000) were used. Mouse genotyping was performed on tail DNA. Neo PCR primer sequences were the following: NeoF 5′-CCGGCCGCTTGGGTGGAGAGG and NeoR 5′-products on 300 bp (targeted allele), and TRPV1Rs (VRF1 5′-CATGGCCAGTGAGAACACCATGG and VRR2 5′-AGCCTTTTGTTCTTGGCTTCTCCT) products on 150 bp (wild type allele). Amplification reactions were carried out in 25 μl total volume with presence of 1% dimethyl-formamide, 0.2 μM primer each, 0.2 μM dNTP, 1.5 mM MgCl2 (93 °C for 15 s, 58 °C for 15 s, 72 °C for 45 s, for 30 cycles). Amplification products were analyzed by agarose gel electrophoresis on 1.2% agarose gels. An example for the results of genotyping of a litter is shown in Fig. 1.
The animals were deeply anaesthetized with isoflurane followed by decapitation. After opening the skull, the brain was quickly removed and immersed into ice-cold cutting solution containing (in mM: NaCl 126, KCl 2.5, NaHCO3 26, CaCl2 0.5, MgCl2 5, NaH2PO4 1.25, glucose 10) bubbled with 95% O2/5% CO2 (carbogen gas). Thick horizontal slices (300–350 μm from mice) were prepared using a Leica VT1000S Vibratome. The slices were stored in an interface type chamber containing ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose) at room temperature for at least 1 h before recording.
Whole-cell patch-clamp recordings were obtained at 34–36 °C from granule cells in the dentate gyrus visualized by infrared videomicroscopy (Versascope, Marton Electronics, Canoga Park, CA). Patch electrodes were pulled from borosilicate glass capillaries with an inner filament (1.5 mm o.d., 1.12 mm i.d.; Hilgenberg, Germany) using a Sutter P-87 puller. Electrodes (∼3–6 MΩ) were filled with a solution containing (in mM) 80 CsCl, 60 Cs-gluconate, 3 NaCl, 1 MgCl2, 10 HEPES, 2 Mg-ATP, and 5 QX-314 (pH 7.2–7.3 adjusted with CsOH; osmolarity 275–290 mOsm). Excitatory postsynaptic currents (EPSCs) were recorded at a holding potential of −65 mV. Slices were perfused with ACSF containing 70–100 μM picrotoxin to block inhibitory neurotransmission. The solution was bubbled with carbogen gas at room temperature and perfused at a flow rate of 2–3 ml/min in a submerged type chamber. To evoke EPSCs, the stimulating electrode was placed in the stratum moleculare of the dentate gyrus. Pairs of electrical stimuli separated by 50 ms were delivered via a theta glass pipette (Sutter Instrument Company, Novato, CA) filled with ACSF at 0.1 Hz using a Supertech timer and isolator (Supertech Ltd., Pécs, Hungary, http://www.superte.ch). Access resistances (between 4 and 18 MΩ, compensated 65–70%) were frequently monitored and remained constant (±20%) during the period of analysis. Signals were recorded with an Axopatch 200B (Molecular Devices, Sunnyvale, CA), filtered at 2 kHz, digitized at 6 kHz (National Instruments PCI-6024E A/D board, Austin, TX), and analyzed off-line with the EVAN program (courtesy of Prof. I. Mody, UCLA, CA).
The drugs were perfused until the maximal effect was reached (usually 3–4 min). The effect of drugs on evoked EPSCs was calculated as follows: control EPSC amplitudes in a 2–3 min time window were compared to those measured after 5–6 min drug application for the same period of time. Only those experiments were included that had stable amplitudes at least for 10 min before drug application. The paired-pulse ratio was calculated from the mean amplitude of the second EPSCs divided by the mean amplitude of the first EPSCs. For spontaneously occurring EPSCs, the amplitude and the inter-event interval for individual events were calculated and medians of their distributions were compared before and after 5 min of capsaicin application. After each experiment, the tubing made of Teflon was washed with ethanol for 10 min and with ACSF for 15 min. For comparison of data, Wilcoxon matched pairs test or Mann–Whitney U-test were used in STATISTICA 6.1 (Statsoft, Inc., Tulsa, OK). Data are presented as mean ±S.E.M.
Picrotoxin was purchased from Sigma–Aldrich, while (E)-capsaicin and capsazepine were obtained from Tocris. Both drugs were dissolved in DMSO giving a 100 mM stock solution, which were stored at 4 °C.
3
Results
The effects of the prototypic TRPV1R agonist capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare were measured in dentate granule cells of wild type mice and TRPV1R knockouts. Similar to what we found earlier (Hájos and Freund, 2002), bath application of 10 μM capsaicin significantly reduced the amplitude of EPSCs (by 36.6 ± 6.1% of control) in wild type mice (control: 143.8 ± 19.3 pA; capsaicin: 88.6 ± 12.1 pA; n = 6; p < 0.02; Fig. 2A and B). In TRPV1R knockout mice, a similar significant reduction was observed after capsaicin application, the amplitude of EPSCs was suppressed by 31.6 ± 4.1% of control (control: 146.9 ± 34.2 pA; capsaicin: 103.8 ± 26.9 pA; n = 6; p < 0.02; Fig. 2A and B). The inhibitory effect of capsaicin on the amplitude of EPSCs was indistinguishable in wild type mice and TRPV1R knockouts (p > 0.1).
Next, we tested the effect of 10 μM capsazepine, a TRPV1R antagonist, on excitatory synapses. We found that bath application of this drug also significantly reduced the EPSC amplitude (by 34.9 ± 5.4% of control) in wild type animals (control: 165.1 ± 35.5 pA, capsazepine: 112.4 ± 28.2 pA; n = 5; p < 0.05; Fig. 3A and B). When we co-applied 10 μM capsaicin together with 10 μM capsazepine, the amplitude of evoked EPSCs was similarly decreased (by 33.3 ± 9.4% of control; control: 149.5 ± 21.2 pA; capsaicin + capsazepine: 99.6 ± 19.6 pA; n = 4; p < 0.05). In TRPV1R knockouts, suppression of the EPSC amplitude was comparable to that seen in wild type mice (i.e., by 30.2 ± 1.5% of control after capsazepine treatment; control: 205.2 ± 7.3 pA; capsazepine: 143.2 ± 6.4 pA; n = 4; p < 0.05; Fig. 3A and B). Similarly, the treatment of slices with a mixture of capsaicin and capsazepine reduced the EPSC amplitude by 38.1 ± 6.2% of control (control: 211.8 ± 4.4 pA, capsaicin + capsazepine: 131.3 ± 13.5 pA; n = 3; p < 0.05), just like in the wild types. These results suggest that capsazepine, as well as capsaicin alone can reduce the amplitude of EPSCs independent of TRPV1Rs, and their effects are not additive.
By a comparison of the paired-pulse ratios of evoked EPSCs, we next examined whether capsaicin and capsazepine affect excitatory synapses presynaptically or postsynaptically. If glutamate release is altered, then the paired-pulse ratio should change. If the reduction in EPSC amplitude is not accompanied by changes in the paired-pulse ratio, then the conductivity of glutamate receptors should be modified by the drug treatment. Therefore, we first investigated the effect of capsaicin on paired-pulse ratio in wild type mice and TRPV1R knockouts. After drug application, the ratio significantly increased to 121.3 ± 3.2% of control in wild types and to 132.5 ± 9.9% of control in knockouts (n = 6 each, p < 0.02). Comparable to these findings, capsazepine treatment also caused a significant increase in the paired-pulse ratio both in wild types (129.7 ± 10.1%; n = 5; p < 0.05) and knockouts (126.4 ± 8.8; n = 4; p < 0.05). Thus, the effects of both capsaicin and capsazepine appear to be presynaptic, reducing glutamate release from excitatory terminals both in wild type and TRPV1R knockout mice.
In further experiments, we investigated how capsaicin alters the properties of spontaneous EPSCs (sEPSCs). In wild type mice, bath application of capsaicin significantly increased the occurrence of spontaneous events (i.e., reduced the inter-event interval by 32.5 ± 11.8% of control, control: 0.43 ± 0.23 s; capsaicin: 0.2 ± 0.05 s; n = 5; p < 0.04, Fig. 4A and B) without changing their amplitude (control: 16.6 ± 2.1 pA; capsaicin: 16.1 ± 2.7 pA; n = 5; p > 0.1; Fig. 4A and B). Similarly, capsaicin also elevated the frequency of sEPSCs in TRPV1R knockouts, since the inter-event interval was reduced by 29.3 ± 7.4% of control (control: 0.16 ± 0.04 s; capsaicin: 0.12 ± 0.04 s; n = 6; p < 0.02; Fig. 4A and B). Similar to those observed in wild types, the amplitude of synaptic events did not change (control: 19.2 ± 3.6 pA; capsaicin: 17.7 ± 3.3 pA; n = 6; p > 0.1; Fig. 4A and B). The comparison of the decrease in the inter-event interval of sEPSCs between wild type mice and TRPV1R knockouts showed no difference (p > 0.1). These results provided further evidence that capsaicin affected synaptic glutamate release in wild type and TRPV1R knockout mice to a similar degree, in a similar manner.
4
Discussion
Electrophysiological data presented here strongly suggest that capsaicin actions on excitatory synaptic transmission are not mediated by TRPV1Rs, at least in the dentate gyrus. Our previous observations already raised this possibility (Hájos and Freund, 2002), as we have shown that the suppression of the amplitude of EPSCs after the second application of capsaicin was indistinguishable from that seen after the first application. This observation was not consistent with the known desensitization properties of TRPV1Rs upon repeated capsaicin application (Dray et al., 1989; Docherty et al., 1991; Caterina et al., 1997). Our results seem to contradict those pharmacological data, where the effect of capsaicin on synaptic transmission has been found to be fully blocked by antagonists specific for TRPV1Rs (e.g., capsazepine or iodo-resiniferatoxin) (Al-Hayani et al., 2001; Marinelli et al., 2003). Here we found that, in adult mice, capsazepine also effectively reduced the amplitude of EPSCs, similar to that seen after capsaicin application. In line with these data, a study by Kofalvi et al. (2003) has shown that glutamate release from synaptosomes prepared from the hippocampus of adult rats could be significantly suppressed by capsazepine. These findings are in contrast with our published results (Hájos and Freund, 2002), where capsazepine could antagonize the effect of capsaicin on EPSC amplitude recorded in slices from rats of P15-22. We repeated the experiments with capsazepine in young rats and found that this drug on its own could substantially enhance the amplitude of EPSCs (unpublished observations), which is in sharp contrast observed in adult animals (present study; Kofalvi et al., 2003). Thus, it seems likely that during development the molecular target of capsazepine changes its effect on synaptic transmission, or the binding site(s) of capsazepine might alter. We therefore propose that, in the hippocampus of young rats, the suppression of EPSC amplitude by capsaicin is counterbalanced by the enhancement caused by capsazepine, therefore no reduction in glutamate release can be observed.
As to the presynaptic mechanism of capsaicin actions, a recent study showed that iodo-resiniferatoxin as well as capsaicin (both applied in μM concentrations) could markedly reduce the high K+-induced Ca2+ entry (Kofalvi et al., 2006). Since transmitter release is highly sensitive to Ca2+ entry, one might assume that glutamate release from excitatory terminals in the dentate gyrus could be affected with a similar mechanism. In the present study we found that capsaicin, capsazepine, or co-application of the two, reduce the amplitude of EPSCs to a similar extent, suggesting that capsazepine might also decrease Ca2+ entry at the same site, where capsaicin acts.
Data from other laboratories, as well as our own results, showed that capsaicin could reduce the amplitude of evoked EPSCs, while it increased the frequency of spontaneous EPSCs without changing their amplitude (Hájos and Freund, 2002; Marinelli et al., 2002, 2003). These unconventional effects seem to suggest that the target molecule of capsaicin regulating glutamatergic transmission could be located at the presynaptic axon terminals, an assumption that is also supported by the observed increase in the paired-pulse ratio (present study). If capsaicin modulated postsynaptic glutamate receptors, the amplitude of sEPSCs should have also been altered, which was not the case (present study; Marinelli et al., 2003). The question arises how capsaicin could reduce the amplitude of evoked EPSCs, while in the same time increases the frequency of sEPSCs. One explanation might be that if capsaicin triggers release of retrograde messengers from postsynaptic neurons that directly promote fusion of vesicles irrespective of Ca2+ concentration within the terminals, this could enhance the action potential-independent glutamate release (i.e., the majority of sEPSCs under our circumstances). Although TRPV1Rs have been shown to be located in the dendrites and somata of hippocampal principal cells (Cristino et al., 2006), these receptors are unlikely to be involved in the enhancement of sEPSC frequency, since the same effect was observed in the TRPV1R knockout mice. Further studies should clarify the possible mechanisms underlying the capscaicin-induced changes in excitatory neurotransmission.
In summary, our data presented here suggest that TRPV1Rs may not be the target molecules for capsaicin regulating glutamatergic synapses in the hippocampal network, although they might have a function during pathological conditions like fever or osmotic changes in extracellular space (Caterina et al., 1997; Gavva et al., 2007; Liu et al., 2007).

excitatory synapses
granule cell
dentate gyrus
glutamate
transmitter release
brain slices


Aptamers as molecular recognition elements for electrical nanobiosensors

Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors. This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors.
Introduction
With the completion of the Human Genome Project, we have a better understanding of disease-related biomaterials such as DNA, RNA, proteins and small biological molecules. Such knowledge can impact strongly on human health, because the identification of a disease-specific marker at an early stage of a disease can greatly improve the clinical success rate, and improve disease-related mortality. For early detection of disease, it is necessary to locate minute amounts of disease-related proteins, peptides or other small molecules in a patient’s bloodstream or body fluid. Nanotechnology combined with biotechnology is expected to provide paradigm-breaking solutions in this area, as these technologies are extremely sensitive [1]. Already, nanoscale materials such as quantum dots [2] and metallic nanoparticles [3, 4], as well as nanodevices such as the nanocantilever [5], and nano field-effect transistors (FETs) [6–9] show potential for detecting tiny molecular signals.
In this review, we concentrate on nanoscale electrical biosensors with aptamers as molecular recognition elements. Biosensors are devices that can be used to detect the presence of a target analyte. As shown in Fig. 1, biosensors are built up from a sample delivery component, a molecular recognition subsystem, a transducer changing the binding or reaction of target molecules into a measurable physical signal (mass, charge, heat or light) and a signal-processing unit. We are particularly interested here in electrical transducers, because they provide the most promising solutions for point-of-care disease detection. Biosensors using electrical transducers are rapid and convenient, and they do not require heavy optical instruments or expensive measuring devices.
Fig. 1A biosensor. MIP molecularly imprinted polymer
Aptamers as molecular recognition elements
For the highly sensitive detection of a target analyte, the selectivity of the recognition element is vital in sensor performance. Until recently, immunosensors using antibody–antigen recognition were employed for biodetection. However, highly sensitive and stable biosensors may use different recognition elements. In the 1980s, functional nucleic acids that can specifically bind and regulate viral or celluar proteins with high affinity were discovered [10]. The major breakthrough for functional nucleic acids occurred in 1990. First, the presence of small functional RNA molecules (TAR aptamers) that can inhibit viral replication [11] was revealed; they can effectively bind and inhibit the activity of viral protein, thereby raising the possibility that they may be used as therapeutic agents. In the second study in 1990, Tuerk and Gold [12] established the in vitro screening process termed "systematic evolution of ligand by exponential enrichment" (SELEX), to identify the RNA ligands with affinity for T4 DNA polymerase and various organic dyes [12]. Such nucleic acids were termed "aptamers" [from the Latin aptus (fit or appropriate)] by Ellington and Szostak [13].
The SELEX process is a technique for screening a very large library of oligonucleotides with random sequences by iterative cycles of selection and amplification. Figure 2 shows a schematic diagram of the SELEX process [14]. The starting pool must be large enough to generate a high probability of producing a desired aptamer (normally around 1015 different sequences). The initial library of random-sequence oligonucleotides, which is obtained through combinatorial chemical synthesis, is incubated with a target of interest. Oligonucleotides showing affinity for the target are partitioned by affinity chromatography or filtration, and then amplified by PCR (for DNA libraries) or reverse transcription PCR (for RNA libraries) to create a new pool enriched in those oligonucleotides having a higher affinity for the target. As this cycle of selection and amplification is repeated, the abundance of the high-affinity oligonucleotides increases exponentially. Negative selection and counterselection are often employed in order to remove aptamers which bind to supports and molecules similar to the target, respectively. Typically after eight to 15 cycles, cloning and sequencing of the enriched library are carried out, revealing the sequence of oligonucleotides highly specific to the target. Once the sequence information has been obtained, the desired aptamer can be readily produced by chemical synthesis. For a given target molecule, DNA aptamers and RNA aptamers can be generated. However, they are quite different in sequence and three-dimensional structure since DNA lacks the 2′ hydroxyl group of the RNA. For example, both the DNA version of the RNA aptamer for ATP and the RNA version of the DNA aptamer for ATP do not recognize ATP [15].
Fig. 2The systematic evolution of ligand by exponential enrichment (SELEX) process [14]
In Table 1, we summarize the advantages of aptamers over antibodies and the yellow-colored rows detail special advantages of aptamers as recognition elements [16]. First, aptamers possess high target affinity. It has been generally recognized that aptamer affinity is comparable to or even higher than that of antibodies, even though this is not always true. Unlike antibodies, aptamers can distinguish between targets even if the targets are in the same family. For example, anti-caffeine aptamers have lower affinity (by a factor of 104) for theophylline, despite the two compounds differing by only a single methyl group [17]. Because aptamers are selected using an in vitro selection process (SELEX), they are cost-effective, have uniform activity, and antitoxin aptamers can be readily synthesized. Antitoxin antibodies are more difficult to prepare, since they are cultured in animal cells. The immobilization of the recognition element can be crucial in biosensors, and this is easier with aptamers than with antibodies, because chemical modification of nucleic acids is simple and straightforward compared with that of antibodies. One of the biggest advantages of aptamers lies in their high stability. Being nucleic acids, aptamers are highly stable, and therefore have unlimited shelf lives. Also, because aptamers can undergo reversible changes of conformation with variations in temperature or salt concentration, aptamer-based sensors are potentially recyclable. The major limitation of aptamers (especially RNA aptamers) as molecular recognition elements is degradation by nucleases. However, it was shown that the problem can be overcome through chemical modifications of the sugar at the 2′-position [18] or use of mirror-image analogs [19]. Recently chemically modified nucleic acid aptamers for in vitro selections have been reviewed [20].
Table 1Advantages of aptamers over antibodies [16]
As aptamers show high target affinity and high target specificity, they can be used as therapeutic agents, in affinity columns and as recognition elements in sensors detecting a wide range of targets. There are virtually no limits of targets for aptamers; small molecules [14], proteins [15] and antibiotics [21]. The first use of an aptamer as a molecular recognition element in sensors was reported in 1996, when fluorescent-tagged aptamers were employed for optical detection of human neutrophil elastase [22]. Since then, various transducers employing aptamers for recognition have been reported. These include the quartz crystal microbalance [23], the surface plasmon resonance technique [24], evanescent-wave-induced fluorescence [25], microcantilevers [26], electrochemical sensors [27–35] and FETs fabricated with single-walled carbon nanotubes (SWNTs) [36, 37]. In this review, we summarize recent advances in electrochemical and FET types of aptamer sensors, focusing on the high sensitivity and recyclability of the aptamer-containing devices.
Electrical aptamer sensors
Electrochemical aptamer sensors
Typical electrochemical sensors operate by reacting with an analyte of interest and producing an electrical signal proportional to the analyte concentration. Many papers have reported the fabrication of electrochemical aptamer sensors where specific binding of an analyte to an aptamer on an electrode can produce an electrical signal [27–35]. Some authors use sandwich assays, employing a secondary aptamer for signal enhancement [28, 29]. However, a technique that is label-free and fast in response is favored especially for electrical types of biosensors. A difficulty with sandwich assays has recently been removed by Xiao et al. [31], who developed a “signal-on” architecture. Earlier electrochemical aptamer sensors used a “signal-off” architecture [30], in which the binding of the target analyte resulted in a decrease in the signal. In such cases, no more than 100% of the signal can be suppressed by target binding, and hence the dynamic range of the sensor is limited. Also, it is difficult to distinguish a real binding event from a false signal originating from contaminants or sensor degradation. To fabricate the signal-on electrochemical aptamer sensor, Xiao et al. introduced a short DNA sequence tagged with methylene blue (MB) that can hybridize with an aptamer. A single-stranded DNA aptamer, which binds to thrombin, was chosen as a model system. Thrombin is a proteolytic enzyme which facilitates blood clotting by converting fibrinogen into fibrin and is also regarded as a tumor marker in the diagnosis of pulmonary metastasis [38]. Figure 3 shows a schematic diagram of the signal-on aptamer sensor. As shown, the sensor is constructed by immobilizing a thiolated thrombin aptamer to a gold electrode, and an MB-tagged partially complementary DNA is added to form a double-stranded complex. In this configuration, owing to the large persistence length of double-stranded DNA, the MB moiety is far away from the sensor surface, and only a small Faradaic current is observed. The persistence length, defined as the distance over which the direction of a polymer segment persists, provides a measure of elasticity in polymer chains. In the case of double-stranded DNA, the persistence length is about 50 nm, or 150 bp [39]. Since the thrombin aptamer is much shorter than 150 bp [40] (the recognition part is only 15 bp in length), the complex would be in a “standing” position. When thrombin is introduced to such a complex, the thrombin aptamer binds to thrombin, and the MB-tagged single-stranded DNA approaches the electrode surface, improving the electron transfer with an increase of current. The persistence length of a single-stranded DNA is 1.5 nm in 2 M NaCl solution, and 3 nm in 25 mM NaCl solution [41].
Fig. 3Thrombin binding with an engineered aptamer. MB methylene blue. (Reprinted with permission from [31], copyright 2005 American Chemical Society)
In the work of Xiao et al., a threefold signal gain was observed with 260 nM thrombin, while only a 40% signal decrease was observed using a signal-off sensor. The detection limit of the sensor thus rose to 3 nM as shown in Fig. 4. Such a high sensitivity is comparable even with that given by sensors using amplification by labeling [28, 29] or optical sensors using evanescent-wave-induced fluorescence anisotropy [25]. Table 2 summarizes the electrochemical aptamer sensors available for thrombin.
Fig. 4Electrochemical signal from the signal-on aptamer sensor. (Reprinted with permission from [31], copyright 2005 American Chemical Society)Table 2The detection limits of an electrochemical aptamer sensor for thrombin Detection limitCharacteristicsReference13 nMUses an MB-tagged "holding" DNA sequence[31]280 nM/3.5 nMPeroxidase-labeled thrombin gives an 80 nM detection limit. Biotin-labeled aptamer with horse radish peroxidase labeled streptavidin gives a detection limit of 3.5 nM[27]31 μMSandwich assay using two aptamers with different binding sites. Secondary aptamer labeled with glucose dehydrogenase[28]410 nMSame as for 3, but pyrroquinoline quinine glucose dehydrogenase is used for labeling[29]56.4 nMMB-tagged thrombin aptamer (signal-off sensor)[30]60.1–0.15 nMThrombin aptamer with ferrocene moiety (square-wave voltametry or chronopotetiometry used for the measurement)[32]711 nMMB-intercalated thrombin aptamer[25]MB methylene blue
Owing to the unique three-dimensional structure of an aptamer, it is not even necessary to use "holding" DNA sequences. Baker et al. [34] reported the rapid, label-free detection of cocaine in both adulterated samples and biological fluid. The cocaine aptamer engineered by Stojanovic et al. [42] was used in this work. This aptamer is thought to form a partially unfolded structure, with only one of its three double-stranded stems intact, when target molecules are absent. In the presence of target, the aptamer folds into the cocaine-binding three-way junction structure of Fig. 5a. With this unique approach, Baker et al. reported the detection of 500 μM cocaine in biological fluid, even when contaminants were present. The ability to detect cocaine in undiluted biological fluids (blood, for example) is crucial for simple forensic detection technology. The binding of small molecules to an aptamer is highly reversible, so it is possible to regenerate the sensor. Baker et al. [34] could regenerate the sensor to a degree of 99% by simply washing a used sensor with buffer.
Fig. 5a Binding of coccaine with an MB-tagged aptamer. b Binding of platelet-derived growth factor (PDGF) with an MB-tagged aptamer. (a Reprinted with permission from [34], copyright 2006 American Chemical Society; b reprinted with permission from [35], copyright 2007 American Chemical Society)
Regeneration of an aptamer sensor is also possible after protein binding. Lai et al. [35] reported the detection of picomolar levels of platelet-derived growth factor (PDGF) directly in blood serum. PDGF is a protein that regulates cell growth and division, and is very important for blood vessel formation (angiogenesis). While generally undetectable in normal cells, PDGF is overexpressed in a variety of disease such as atherosclerosis, and human tumors including sarcomas and glioblastomas [14, 43]. As shown in Fig. 5b, specific binding of PDGF to the aptamer decreases the distance between MB and the electrode and consequently increases the electrical signal. The sensor could be also regenerated by 4-min incubation in 10% (w/v) sodium dodecyl sulfate.
In this section, we have shown that engineered aptamers, employed as recognition elements in electrochemical transducers, can greatly improve sensor performance. It is an advantage of aptamers over antibodies that aptamers can be engineered to either enhance or activate signals. Recently, aptazymes (aptamers with catalytic activities) have been reported, which combine an enzymatic role with a recognition function [44]. Carefully designed aptazymes can be developed as sensors that do not require labeling. An electronic (electrochemical) sensor approach with carefully designed aptamers offer potential for highly sensitive, selective and reusable sensor platforms.
Aptamer sensors with carbon nanotube field-effect transistor transducers
Since the first report on the biosensing ability of Si nanowire in 2001 [45] and the development of the chemical sensor operation of carbon nanotubes in 2000 [46], extensive research efforts have been initiated aimed at developing nanotube-based or nanowire-based sensors. Owing to the extremely large surface-to-volume ratio of one-dimensional nanostructures, it is possible to develop sensors with exquisite sensitivity. This is especially true in the case of carbon nanotubes, where all constituent atoms are at the surface. Any small disturbance of or adsorption to the surface could result in a large change in electrical conductance. Already, biosensors fabricated with semiconducting nanowires or nanotubes have been used as sensors that can detect a single virus [6], small molecules [47] or minute amounts of proteins in solution [7–9].
FET sensors measure a change in the surface potential occurring during the binding between an analyte and a recognition element. Figure 6 shows a schematic diagram of the working principle of carbon-nanotube-based FET sensors. FET sensors “feel” the charges when target molecules bind with probe molecules immobilized on the sensor surfaces. Such an effect is often referred to as an “electrostatic gating effect” since the bound target molecules behave as an additional gate electrode. In the case of carbon nanotubes, SWNTs behave as p-type semiconductors in ambient atmosphere. In p-type FETs, positive gate voltage decreases the conductance (depletion) and negative gate voltages increase the conductance (accumulation) from the device. Therefore, if positively charged molecules bind with probe molecules on SWNT-FETs, this event appears as a decrease of conductance, while binding of negatively charged molecules appears as an increase of conductance in SWNT-FETs as denoted in Fig. 6.
Fig. 6The working principle of field-effect transistor (FET) sensors based on carbon nanotubes
Using FETs for immunosensing was first proposed by Shenk [48] in 1978. It was suggested that FETs could be used to detect surface polarization owing to the formation of an antibody and an antigen. This concept failed, however, because small ions in the solution caused a screening effect inhibiting detection of the changes [49]. Figure 7 shows the charged semiconductor surface in solution. As shown in Fig. 7, when a charged surface (this might be a metal electrode, a semiconductor or a charged dielectric) is brought into contact with an ionic solution, the surface charge potential is dissipated by redistribution of counter ions in the solution. The domains where the variation of electric potential occurs are called electrical double layers, and the depth of an electrical double layer can be determined by calculating the Debye screening length. The Debye screening length is the scale over which mobile charge carriers screen the electric field. In an electrolyte, the Debye screening length can be defined as follows:
Fig. 7Electrical double layer at the sensor surface
Here the Bjerrum length lB is defined as
and is 0.7 nm at standard pressure and temperature. kB is the Boltzmann constant, k is defined as 1/4πɛ0, ρi is the number density of ions and the zi values are valencies of various ions.
The Debye screening length, therefore, is proportional to the inverse square root of the ion number density, i.e., to ionic strength. For pure water, the Debye screening length is about 1 μm, while it is only 0.3 nm in 1 M NaCl solutions. In 1 M NaCl solution, therefore, two ions or charged species separated by 1 nm do not interact with each other. If we consider the sensor surface, any interactions occurring outside the Debye screening length cannot be detected using FET sensors. The biggest disadvantage of FET sensors is that they may not work with blood samples or body fluids, because the ionic strength of such physiological solutions is about 150 mM, which yields a Debye screening length of about 1 nm.
Until recently, FET-type sensors have employed an antibody as the recognition receptor. However, the typical size of an antibody is about 10–15 nm [50, 51]. In that case, the binding of an antigen with an antibody occurs far outside the Debye screening length in physiological solutions. Molecular recognition elements with high specificity, selectivity and small sizes are essential for applications of FET-type sensors. In this respect, aptamers are highly suitable. Figure 8 shows the typical size ranges of antibodies and aptamers [52]. Because aptamers are mostly short nucleic acids ranging from 10 to 60 bp in length, the binding of target molecules with aptamers can occur inside the electrical double layer, even in 20–50 mM salt solutions.
Fig. 8Size comparison of an aptamer with an antibody. IgG immunoglobulin G. (Reprinted from [52], copyright 2006 Elsevier)
Such a biggest merit of aptamers in FET sensors was first reported by So et al. [36] using a carbon nanotube biosensor with thrombin aptamers as molecular recognition elements. They also demonstrated the sensitivity and selectivity of aptamers in SWNT-FETs. Anti-thrombin aptamers (5′-GGT TGG TGT GGT TGG-NH2-3′) were immobilized onto the surface of the SWNT-FET, fabricated using a patterned growth technique, with carbonyl diimidazole–Tween 20 as the linking molecules (Fig. 9a). The authors observed an abrupt decrease in conductance upon reaction with thrombin, while almost no changes were observed with elastase (Fig. 9b). Elastase is a member of the thrombin family and has almost the same molecular weight and isoelectric point as thrombin. Moreover, the sensor could be regenerated by simple washing with 6 M guanidine hydrochloride solution. The authors regenerated the sensor more than five times without significant loss of activity. As shown in Fig. 9c, the authors measured the sensitivities of SWNT-FET sensors with aptamers as recognition elements. Before each measurement, the sensor was "reset" by washing it with guanidine hydrochloride solution. The lowest detection limit was around 10 nM thrombin, and the sensor signal was saturated at about 100 nM thrombin.
Fig. 9a Single-walled carbon nanotube (SWNT) FET sensor with aptamers as molecular recognition elements. b Selectivity of an anti-thrombin aptamer-functionalized SWNT-FET. c Sensitivity of an anti-thrombin aptamer-functionalized SWNT-FET. The sensor was reset by washing it with 6 M guanidine hydrochloride solution before each measurement. CDI carbonyl diimidazole. (Reprinted with permission from [36], copyright 2005 American Chemical Society)
Maehashi et al. [37] have recently addressed the size issue of recognition elements in FET-based sensors. In their work, immunoglobulin E (IgE) aptamer (5′-NH2-GCG CGG GGC ACG TTT ATC CGT CCC TCC TAG TGG CGT GCC CCG CGC-3′) and monoclonal anti-IgE antibody (IgE-mAb) were immobilized on carbon nanotubes, with the aid of the linking molecule 1-pyrenebutanoic acid succinimidyl ester. Figure 10 a and b shows the responses of the aptamer-functionalized and antibody-functionalized sensors, respectively, when they were exposed to known concentrations of target IgE in 10 mM phosphate-buffered saline (PBS). As shown in Fig. 10, the aptamer-functionalized sensor exhibited small changes with only 250 pM target protein molecules, while a negligible change in conductance was observed in the antibody-funtionalized sensor even with 140 nM IgE. In 10 mM PBS, the Debye screening length would be roughly 3 nm, and the binding of IgE with the aptamer would be safely inside the electrical double layer. With IgE-mAb, however, there is little hope that binding can be detected in the buffer solution used for the experiment, because the antibody molecule is about 10 nm long [53].
Fig. 10The sensor signal measured from a an aptamer-functionalized SWNT-FET and b an antibody-functionalized SWNT-FET. IgE immunoglobulin E, PBS phosphate-buffred saline. (Reprinted with permission from [37], copyright 2007 American Chemical Society)
As discussed, aptamers may allow a wider range of analytes in FET sensors owing to their small size. SWNT-FET sensors with aptamers as recognition elements showed high sensitivity and selectivity, and could be readily regenerated. Highly sensitive carbon nanotube sensors combined with small, economic, highly selective and stable aptamers could provide cost-effective point-of-care testing devices.
Concluding remarks
This review has focused on the advantages of electrochemical and FET sensor types with aptamers as recognition elements. Through use of aptamers for recognition, no labeling is required for electrochemical sensors, and signal-on architecture that is only possible with aptamers has made possible the sensitivity improvements. Also, as pointed out already, sensors can be recycled because of the reversibility of aptamer configurations. Alternatively, bound proteins may be simply washed off, without damaging the aptamers. In immunosensing, by contrast, it is practically impossible to remove bound antigens from antibodies without damaging the antibodies, because both antibodies and antigens are proteins in nature. Aptamers have proved their superiority over antibodies in nano-FET sensors. When no signal can be measured with an antibody–antigen pair because of the large size of the antibody, the binding of small aptamers to various targets can occur inside the electrical double layer where the nanotube can "feel" the change.
Owing to the rapid aging of society, there is great demand for paradigm-breaking biosensors to detect the onset of disease with speed, convenience and accuracy. In a few more years, electrical nanosensors, with engineered aptamers, will become prominent in the market. They will be small in size, high in sensitivity and competitive in price.

aptamers
nanobiosensors
electrochemical aptamer sensors
field-effect transistor aptamer sensors