Acoustofluidic Micromixing Enabled Hybrid Integrated Colorimetric Sensing, for Rapid Point-of-Care Measurement of Salivary Potassium.

The integration of microfluidics with advanced biosensor technologies offers tremendous advantages such as smaller sample volume requirement and precise handling of samples and reagents, for developing affordable point-of-care testing methodologies that could be used in hospitals for monitoring patients. However, the success and popularity of point-of-care diagnosis lies with the generation of instantaneous and reliable results through in situ tests conducted in a painless, non-invasive manner. This work presents the development of a simple, hybrid integrated optical microfluidic biosensor for rapid detection of analytes in test samples. The proposed biosensor works on the principle of colorimetric optical absorption, wherein samples mixed with suitable chromogenic substrates induce a color change dependent upon the analyte concentration that could then be detected by the absorbance of light in its path length. This optical detection scheme has been hybrid integrated with an acoustofluidic micromixing unit to enable uniform mixing of fluids within the device. As a proof-of-concept, we have demonstrated the real-time application of our biosensor format for the detection of potassium in whole saliva samples. The results show that our lab-on-a-chip technology could provide a useful strategy in biomedical diagnoses for rapid analyte detection towards clinical point-of-care testing applications.

A design for simulating and validating the nuss procedure for the minimally invasive correction of pectus excavatum.

Surgical planners are used to achieve the optimal outcome for a surgery, especially in procedures where a positive aesthetic outcome is the primary goal, such as the Nuss procedure which is a minimally invasive surgery for correcting pectus excavatum (PE)--a congenital chest wall deformity. Although this procedure is routinely performed, the outcome depends mostly on the correct placement of the bar. It would be beneficial if a surgeon had a chance to practice and review possible strategies for placement of the corrective bar and the associated appearance of the chest. Therefore, we propose a strategy for the development and validation of a Nuss procedure surgical trainer and planner.

Stimulatory current at the edge of an inactive conductor in an electric field: role of nonlinear interfacial current-voltage relationship.

Cardiac electric field stimulation is critical for the mechanism of defibrillation. The presence of certain inactive epicardial conductors in the field during defibrillation can decrease the defibrillation threshold. We hypothesized this decrease is due to stimulatory effects of current across the interface between the inactive conductor and the heart during field stimulation. To examine this current and its possible stimulatory effects, we imaged transmittance of indium-tin-oxide (ITO) conductors, tested for indium with X-ray diffraction, created a computer model containing realistic ITO interfacial properties, and optically mapped excitation of rabbit heart during electric field stimulation in the presence of an ITO conductor. Reduction of indium decreased transmittance at the edge facing the anodal shock electrode when trans-interfacial voltage exceeded standard reduction potential. The interfacial current-voltage relationship was nonlinear, producing larger conductances at higher currents. This nonlinearity concentrated the interfacial current near edges in images and in a computer model. The edge current was stimulatory, producing early postshock excitation of rabbit ventricles. Thus, darkening of ITO indicates interfacial current by indium reduction. Interfacial nonlinearity concentrates current near the edge where it can excite the heart. Stimulatory current at edges may account for the reported decrease in defibrillation threshold by inactive conductors.

Myocardial electrical impedance as a predictor of the quality of RF-induced linear lesions.

Production of complete (i.e. continuous and transmural) cardiac lesions by radiofrequency (RF) ablation can cure certain cardiac arrhythmias. However, a predictor of lesion completeness that is reliable and can be measured intraoperatively is needed in order to maximize effectiveness of ablation therapy. Predictors that require membrane excitation or response to stimulation are not always practical. This study tested whether changes of myocardial impedance across the lesion can predict completeness. RF energy was applied epicardially on perfused rabbit ventricles to produce linear lesions that were complete (n = 25) or incomplete (noncontinuous or nontransmural, n = 25). Before and after creation of each lesion, the magnitude and phase of impedance at 1 kHz were measured with a four-electrode epicardial array across the lesion. For 16 of the lesions, the translesion stimulus-excitation delay was also measured. Lesion completeness was evaluated with 2,3,5-triphenyltetrazolium chloride stain. Complete lesions increased resistivity by 26 Omega cm (21% of the preablation value, p = 0.0007, n = 17) when the inactive RF electrode remained on the epicardium during impedance measurements. When the RF electrode was removed during measurements, the rise of resistivity by complete lesions increased to 58 Omega cm (30% of the preablation value, p = 0.022, n = 8). For incomplete lesions, resistivity did not change significantly. Ablation did not significantly alter the phase of impedance. Accuracies of predictions of lesion completeness by the change in resistivity or the change in translesion stimulus-excitation delay were comparable (Youden's index 0.75 and 0.625, respectively, n = 16). Thus, RF ablation increases myocardial resistivity. The resistivity can predict lesion completeness and may provide an alternative to predictors based on excitation.

Translesion stimulus-excitation delay indicates quality of linear lesions produced by radiofrequency ablation in rabbit hearts.

Failure of cardiac antiarrhythmic ablation to block action potential conduction produces poor outcomes which lead to repeat procedures. To overcome this, an intraoperative index of the quality of an ablation lesion is needed. We hypothesized that a rise in the translesion stimulus-excitation delay (TED) can indicate a continuous, transmural, linear lesion, and that the TED is related to the path length in the viable tissue around the lesion. Rabbit hearts were isolated, perfused with a warm physiological solution and stained with transmembrane potential-sensitive fluorescent dye. Radiofrequency (RF) ablation was performed on ventricular epicardium with a vacuum-assisted coagulation device to produce either a complete or incomplete lesion. Complete lesions were both transmural and continuous. Incomplete lesions were noncontinuous or nontransmural. The TED was determined with bipolar stimulation at one side of the lesion and either a bipolar electrogram at the other side or optical mapping on both sides. Hearts were then stained with tetrazolium chloride and examined histologically to estimate minimum path lengths of viable tissue from the stimulation site to the recording site. Complete lesions increased the TED by factors of 2.6-3.1 (p < 0.05), whereas incomplete lesions did not significantly increase the TED. Larger minimum path lengths were found for cases that had an increased TED. The TED was quantitatively predictable based on a conduction velocity of 0.38-0.49 m s(-1), which is typical of rabbit hearts. The TED significantly increases when a linear lesion is complete, suggesting that an intraoperative measurement of the TED may help to improve ablation lesions and outcomes. Predictability of the TED based on the viable tissue path suggests that quantitative TEDs for clinical lesions may be anticipated provided that the conduction velocity is considered.

Comparison of optical and electrical mapping of fibrillation.

Optical recordings with transmembrane potential (Vm)-sensitive fluorescent dye, or extracellular potential (Ve) recordings are used to map spatiotemporal patterns of cardiac excitation during ventricular fibrillation (VF). While the optical and electrical methods are accepted, there has not been a test of whether they yield equivalent excitation times during VF. Times may differ since previous results indicate optical Vm interrogates deeper than Ve. We tested whether the steepest parts of the downward deflection of the Ve and upward deflection of optical Vm are synchronized during VF. We used simultaneous coepicentral optical and electrical mapping (32 spots, 4 kHz) with translucent indium tin oxide electrodes and a laser scanner on ventricular epicardium. VF was electrically induced in arterially-perfused rabbit hearts stained with di-4-ANEPPS. For both the optical and electrical deflections, maximum magnitudes of the slopes varied over a > 4 fold range, morphologies varied and spatiotemporal distributions were nonuniform. Time differences between the steepest parts of the optical and electrical deflections were typically a few ms. Standard deviations of time differences increased for the deflections that had the smaller slopes, which was only partly due to effects of recording noise as indicated by simulations. For deflections that had slopes ranging from the steepest found at each spot to 1/4 of the steepest, the optical deflections were on average 0.7-1 ms earlier than the Ve deflections. Thus, excitation times during VF measured optically and electrically differ. Considered together with our earlier results indicating that the optical Vm interrogates deeper than Ve, the results suggest that most fibrillatory excitations occur earlier in subsurface tissue than at the heart surface.

Optical mapping of V(m) and Ca(i)(2+) in a model of arrhythmias induced by local catecholamine application in patterned cell cultures.

Catecholamines are known to provoke cardiac arrhythmias, but important aspects such as localization of the arrhythmia source in multicellular tissue and exact ionic mechanisms are not well-known. In this work, a multicellular model of arrhythmias caused by local epinephrine application was developed; V (m) and Ca(i)(2+) changes at the arrhythmia source were measured using fluorescent dyes and high-resolution optical mapping. Cultured strands of neonatal rat myocytes (width approximately 0.4 mm) were produced by patterned growth. Epinephrine (1 micromol/l) was applied over an area of 0.3-0.6 mm via two micropipettes, and strands were stimulated by burst pacing. Local epinephrine application caused triggered arrhythmias with cycle lengths of 202-379 ms and duration of >10 s in 9 out of 16 preparations. Optical V(m) mapping demonstrated that in 78% of cases, the source of arrhythmia was located at the boundary of the locally perfused area. Staining with Ca(i)(2+)-sensitive dye Fluo-4 prevented arrhythmia induction in most cases (85%) likely due to Ca(2+) buffering by the dye. Optical Ca(i)(2+) mapping revealed non-propagated Ca(i)(2+) oscillations at the boundary of the locally perfused area in 45% cases. In conclusion, we developed a new model of catecholamine-dependent arrhythmias allowing mapping of V(m) and Ca(i)(2+) at the arrhythmia source with microscopic resolution. The arrhythmias typically originated from the boundary of the epinephrine-perfused area. The location of the arrhythmia source correlated with localized Ca(i)(2+) oscillations suggesting that arrhythmias were caused by Ca(i)(2+) overload at these locations.

Use of light absorbers to alter optical interrogation with epi-illumination and transillumination in three-dimensional cardiac models.

Cardiac optical mapping currently provides 2-D maps of transmembrane voltage-sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity.

Imaging of cardiac movement using ratiometric and nonratiometric optical mapping: effects of ischemia and 2, 3-butaneodione monoxime.

Transmembrane voltage-sensitive fluorescent dyes are used to study electrical activity in hearts. Green and red fluorescence emissions from di-4-ANEPPS excited with 488 nm light indicate both transmembrane voltage changes and heart movement. We have previously shown that the ratio, green fluorescence divided by red fluorescence, indicates the transmembrane voltage without effects of movement. Here we examine the feasibility of measuring the movement, which is useful for the study of cardiac function, by subtracting this ratiometric signal from the red or green fluorescence signal. The results of this subtraction show tissue movement and its relative changes during cardiac ischemia and perfusion with an electromechanical uncoupling agent. By incorporating the spatial variations in fluorescence intensity from the heart, tissue movement can be qualitatively mapped to examine relative changes, however, with limited ability to quantify absolute displacement. Since these maps are obtained simultaneously with corresponding transmembrane potentials, the method allows study of spatiotemporal cardiac movement patterns and their relationship to the action potential.

Use of translucent indium tin oxide to measure stimulatory effects of a passive conductor during field stimulation of rabbit hearts.

Biomathematical models and experiments have indicated that passive extracellular conductors influence field stimulation. Because metallic conductors prevent optical mapping under the conductor, we have evaluated a passive translucent indium tin oxide (ITO) thin-film conductor to allow mapping of transmembrane potential (V(m)) and stimulatory current under the conductor. A 1-cm ITO disk was patterned photolithographically and positioned between 0.3-cm(2) mesh shock electrodes on the ventricular epicardium of isolated perfused rabbit hearts stained with 4-{2-[6-(dibutylamino)-2-naphthylenal]ethenyl}-1-(3-sulfopropyl)-, hydroxide, inner salt (di-4-ANEPPS). For a 1-A, 10-ms shock during the action potential plateau, optical maps from fluorescence collected using emission ratiometry (excitation at 488 nm and emissions at 510-570 and >590 nm) indicated that the disk altered V(m) by as much as the height of an action potential. DeltaV(m) became more positive near the edge of the disk, where the ITO conductance gradient was parallel to applied current, and more negative near the opposite edge, where the gradient was not parallel to current. For diastolic shocks, the disk expedited membrane excitation at the sites of positive DeltaV(m) in the heart and in a cardiac model with realistic ITO disk surface and interfacial conductances. Optical maps of ITO transmittance and the model indicated that the disk introduced anodal and cathodal stimulatory current at opposite edges of the disk. Thus ITO allows study of the stimulatory effects of a passive conductor in an electric field.

Two-photon excitation of di-4-ANEPPS for optical recording of action potentials in rabbit heart.

Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.

Cardiac optical mapping under a translucent stimulation electrode.

Major effects of stimulation on cardiac transmembrane potentials (Vm) are thought to occur under the electrode, however these have not been optically mapped due to blockage of light by electrodes. Here we optically mapped under translucent indium tin oxide (ITO) electrodes in hearts stained with transmembrane voltage sensitive fluorescent dye, di-4-ANEPPS excited at 488 nm. Emissions in wavelength bands 510-570 nm and >590 nm were similarly affected by changes in ITO transmittance due to electrochemical effects of current at the electrode interface. Dual-wavelength ratiometric mapping with the two emission bands revealed Vm under the electrode during plateau-phase stimulation (220 mA). Changes in Vm were heterogeneous under the electrode, and were anisotropic with larger values along the fiber axis. These results explain early excitation sites for sufficiently strong diastolic stimulation, and agree with theoretical predictions based on summation of anisotropic effects of point stimulation and a linear 3-d cardiac bidomain computer model. The bidomain model and experiments disagree under the edge of the electrode, where modeled Vm is much larger. Thus, changes in Vm under an electrode are anisotropic with greater Vm in the direction parallel to fibers. Nonlinear effects of stimulation in hearts may limit changes in Vm under the electrode edge.

Development of a system for simultaneous 31P NMR and optical transmembrane potential measurement in rabbit hearts.

The aim of this study is to develop a system for the first simultaneous measurements using 31P NMR and optical transmembrane potential-sensitive fluorescence. 31P NMR is used to evaluate 5 metabolic markers (pH, sugar phosphates, phosphocreatine, phospholipid intermediates and ATP). Action potential duration is measured with a transmembrane potential-sensitive fluorescent dye, di-4ANEPPS. The system is intended to correlate the action potentials with the metabolic markers and their changes during the time course of cardiac ischemia. The requirements of this system include fabrication of a NMR probe large enough for rabbit heart experiments, incorporation of light guides for excitation of dye and collection of fluorescence in hearts located within a NMR magnet and with minimal disturbance of the magnetic field. The quality factor (Q) of the probe's coil was measured. Control NMR spectra were then acquired with phosphorus test solution. Further spectra were obtained after addition of the optical elements. Results show the ability to use our new probe to acquire a spectrum in the presence of the optical elements within the magnet, suggesting the possibility that 31P NMR spectroscopy and optical transmembrane potential measurements can be performed simultaneously in hearts.

Spatial localization of cardiac optical mapping with multiphoton excitation.

Depth and radius of regions interrogated by cardiac optical mapping with a laser beam depend on photon travel inside the heart. It would be useful to limit the range of depth and radius interrogated. We modeled the effects of a condensing lens to concentrate laser light at a target depth inside the heart, and near infrared excitation to increase penetration and produce two-photon absorption. A Monte Carlo simulation that incorporated a 0.55-NA lens, and absorption and scattering of 1064- or 488-nm laser light in 3-D cardiac tissue indicated the distribution of excitation fluence inside the tissue. A subsequent simulation incorporating absorption and scattering of transmembrane voltage-sensitive fluorescence (wavelength 669 nm) indicated locations from which fluorescence photons exiting the tissue surface originated. The results indicate that mapping at depths up to 300 microm in hearts can provide significant improvement in localization over existing cardiac optical mapping. The estimated interrogation region is sufficiently small to examine cardiac events at a cellular or subcellular scale and may allow mapping at various depths in the heart.

Emission ratiometry for simultaneous calcium and action potential measurements with coloaded dyes in rabbit hearts: reduction of motion and drift.

INTRODUCTION: Optical measurements of the cardiac calcium transient (Ca) and transmembrane action potential (AP) may be performed simultaneously with emission ratiometry to lessen motion artifacts and photobleaching effects. We examined changes in emission spectrum in perfused rabbit hearts coloaded with Rh237 and a green-emitting Ca dye (Fluo-4 or Oregon Green BAPTA 1) to determine wavelength bands for emission ratiometry and to test whether ratiometry reduces motion artifacts and drift. METHODS AND RESULTS: A 488-nm laser illuminated hearts while a spectrofluorometer collected fluorescence from 489 to 838 nm at 1 kHz. Ratiometry with the Ca- and AP-insensitive emission band 663 to 685 nm (IS) as denominator and the Ca-sensitive band 510 to 532 nm as numerator lessened motion artifacts, which was quantified as a 1.4-fold increase in relative amplitude of the Ca (P < 0.05). Ratiometry with the AP-sensitive band 772 to 794 nm as denominator and the IS as numerator produced a 1.7-fold increase in relative amplitude of the AP (P < 0.05). The ratiometry decreased photobleaching-dependent drift by a factor of 0.6 (P < 0.05) for Ca and 0.45 (P < 0.05) for AP. CONCLUSION: Simultaneous Ca and AP emission ratiometry reduces motion artifacts and drift in hearts with coloaded dyes.

Simultaneous electrical and optical mapping in rabbit hearts.

Local cardiac excitation is measured from either extracellular voltage (Ve) or optical transmembrane voltage (Vm) with fluorescent dye. We used a transparent electrode array and a laser scanner to test whether Ve and coepicentral optical Vm give equivalent excitation times during epicardial pacing and sinus beats. To help explain time differences, we estimated interrogation width/depth ratios for Ve and optical Vm with dipole and Monte Carlo models. Magnitudes of time differences between excitation measured with Ve and optical Vm during pacing were 1.9 +/- 1.9 ms (n= 1,112 recording pairs). Deeper interrogation for optical Vm vs Ve was indicated with sinus beats that contained a transmural propagation component. When pacing produced propagation along epicardial fibers, excitation time measured from optical Vm was later than excitation time measured with epicardial Ve. When pacing produced propagation across epicardial fibers, excitation time measured from optical Vm was earlier than that measured with epicardial Ve. These time differences could be attributed to deeper optical interrogation and fiber rotation with depth in ventricles.