Integration of thin film amorphous silicon photodetector with lab-on-chip for monitoring protein fluorescence in solution and in live microbial cells

Green fluorescent protein (GFP) and other fluorescent protein variants are widely used as powerful tools to tag and monitor complex biological processes at the cellular, tissue, and organism level. A micrometer-scale GFP detection system is demonstrated in which a p-i-n thin-film amorphous silicon light sensitive layer microfabricated on a glass substrate is integrated with an amorphous silicon carbon alloy absorption filter and an ultra-thin PDMS sheet to detect intracellular expression of GFP. GFP fluorescence was detected in aqueous solution in the nM range, and inside living cells (Escherichia coli) expressing GFP in the 10⁶ cell/mL range. The temporal evolution of the integrated fluorescence from promoter-induced GFP production was monitored in E. coli in solution over 12 h using the detection system. This microfabricated thin-film detection system can potentially be developed into an array-based sensing of GFP allowing for high-throughput and multiplexed analysis of biological and biomedical samples.     

A new acridine derivative as a highly selective ‘off-on’ fluorescence chemosensor for Cd in aqueous media

A new acridine fluoroionophore containing two diethanolamine ligands, 4,5-bis(N,N-di (2-hydroxyethyl)iminomethyl)acridine (BHIA), was designed and synthesized based on the fluorophore-spacer-receptor format. And its fluorescent sensing behavior towards metal ions was investigated in buffered aqueous media. The presence of Cd²⁺ induces the formation of a 1:1 ligand/metal complex at neutral pH, which exhibits enhanced emission at 454 nm. The fluorescence intensity is linear with the Cd²⁺ concentration in the range of 1.0 × 10⁻⁶ to 3.0 × 10⁻⁵ M (R = 0.9967). Experimental results show a low interference response towards other metal ions. The selective switch-on fluorescence response of BHIA to Cd²⁺ makes it suitable for sensing of Cd²⁺ in aqueous solution. The detection limit is 1.3 × 10⁻⁷ M. Moreover, the results indicated that BHIA was a reversible chemosensor for Cd²⁺, which makes it attractive for sensing applications.     

A new optochemical chlorine gas sensor based on the application of amphiphilic co-networks as matrices

A sensor for chlorine gas detection, consisting of an amphiphilic polymer co-network with an immobilised oxidation indicator, o-tolidine, is described. Data describing gas sensing properties and long-term stability will be presented. This study focuses on APCN thin films as a matrix for indicator immobilisation.Thin films of poly(2-hydroxyethyl acrylate)-l-polydimethylsiloxane PHEA-l-PDMS were prepared as immobilisation matrices for o-tolidine.We present a simple, non-expensive, but highly sensitive optical sensor for chlorine gas detection. The thin film response is reproducible and irreversible. With our kinetic-optical method rapid response times were achieved. The determination of chlorine is performed on the basis of the oxidation of o-tolidine as the chromogenic reagent to a coloured product which can be monitored at 650 nm. The results reveal a fast response to chlorine gas down to concentrations of 0.01 ppm.     

Comparison of two hyperspectral imaging and two laser-induced fluorescence instruments for the detection of zinc stress and chlorophyll concentration in bahia grass (Paspalum notatum Flugge.)

Bahia grass (Paspalum notatum Flugge.) plants were grown in silica sand and irrigated daily with one of five levels of Zn (0, 0.5, 25, 50, or 100 mg l⁻¹) to determine the effects of the heavy metal on the growth and development of plant canopies. Healthy and stressed plants were measured with two hyperspectral imagers, laser-induced fluorescence spectroscopy (LIFS), and laser-induced fluorescence imaging (LIFI) systems in order to determine if the four handheld remote sensing instruments were equally capable of detecting plant stress and measuring canopy chlorophyll levels in bahia grass. Symptoms of bahia grass plants grown at deficient (0 mg l⁻¹) or toxic (25, 50, or 100 mg l⁻¹) concentrations of Zn were dominated by leaf chlorosis and plant stunting. Leaf fresh weight, leaf dry weight, CO₂ assimilation, total chlorophyll, and leaf thickness followed (+) quadratic models in which control plants (0.5 mg l⁻¹ Zn) exhibited higher responses than plants grown at either deficient or toxic levels of Zn. Normalized difference vegetation index [NDVI=(NIR−Red)/(NIR+Red)] and ratio vegetation index [RVI=R₇₅₀/R₇₀₀, in which R denotes reflectance] values were calculated for calibrated digital images from both hyperspectral imagers. The NDVI and RVI values from both hyperspectral imagers were fit best by (+) quadratic models when treatments were constrained between 0 and 100 mg l⁻¹ Zn, but were fit best by linear regression models with (−) slopes when treatments were constrained between 0.5 and 100 mg l⁻¹ Zn. Furthermore, both NDVI and RVI algorithms were effective in predicting the concentrations of chlorophyll in canopies of bahia grass grown at the various levels of Zn. In contrast, red/far-red (R/FR) fluorescence ratios estimated from leaf fluorescence values measured with the LIFS and LIFI instruments were fit best by (−) quadratic models when treatments were constrained between 0 and 100 mg l⁻¹ Zn, but were fit best by linear regression models with (+) slopes when treatments were constrained between 0.5 and 100 mg l⁻¹ Zn. A series of regression analyses were conducted among plant biometric, biochemical, and leaf anatomical parameters (treated as independent variables) and the remote sensing algorithms, NDVI, RVI, blue/green (BL/GR), and R/FR (treated as dependant variables). In general, residuals were significantly higher for NDVI and RVI models compared to the BL/GR and R/FR models indicating that the NDVI and RVI algorithms were able to measure total chlorophyll and plant biomass more accurately than the BL/GR and R/FR algorithms. However, unique capabilities of LIFS and LIFI instruments continue to argue for the development of laser-induced fluorescence remote sensing technologies.     

Cu chemosensing behaviour of self-organized micro-array structures of a donor-acceptor bichromophoric compound anchored onto Ag nanoisland films

This work reports on the Cu²⁺ chemosensing behaviour of self-organized micro-array structures of a novel donor-acceptor bichromophoric compound anchored onto Ag nanoisland films. The system exhibits quenching of the fluorescence in the presence of Cu²⁺ ions, with detection range extending from 2 × 10⁻⁸ M up to 3 × 10⁻⁶ M and limit of detection (LOD) of 8 × 10⁻⁹ M. The quenching of fluorescence is accompanied by a quenching of SERS signal from the metal-organic structure, which is consistent with an electron transfer between the copper cation and the organic moiety. The self-organization property of the sensing complexes into micrometric arrays offers great potential for miniaturization and future development of Cu²⁺ detection systems based on real-time observation of fluorescence or SERS quenching by fluorescence microscopy or microRaman spectroscopy.     

Estimation and comparison of evapotranspiration from MODIS and AVHRR sensors for clear sky days over the Southern Great Plains

Evapotranspiration (ET) cannot be measured directly from satellite observations but remote sensing can provide a reasonably good estimate of evaporative fraction (EF), defined as the ratio of ET and available radiant energy. It is feasible to estimate EF using a contextual interpretation of radiometric surface temperature (T_o) and normalized vegetation index (NDVI) from multiple satellites. Recent studies have successfully estimated net radiation (R_n) over large heterogeneous areas for clear sky days using only remote sensing observations. With distributed maps of EF and R_n, it is now possible to explore the feasibility and robustness of ET estimation from multiple satellites. Here we present the results of an extensive inter-comparison of spatially distributed ET and related variables (NDVI, T_o, EF and R_n) derived from MODIS and AVHRR sensors onboard EOS Terra, NOAA14 and NOAA16 satellites respectively. Our results show that although, NDVI and T_o differ with the sensor response functions and overpass times, contextual space of NDVI-T_o diagram gives comparable estimates of EF. The utility of different sensors is demonstrated by validating the estimated ET results to ground flux stations over the Southern Great Plains with a root mean square error of 53, 51 and 56.24 Wm^− 2, and a correlation of 0.84, 0.79 and 0.77 from MODIS, NOAA16 and NOAA14 sensors respectively.     

Comparison of ERS-2 SAR and Landsat TM imagery for monitoring agricultural crop and soil conditions

Studies over the past 25 years have shown that measurements of surface reflectance and temperature (termed optical remote sensing) are useful for monitoring crop and soil conditions. Far less attention has been given to the use of radar imagery, even though synthetic aperture radar (SAR) systems have the advantages of cloud penetration, all-weather coverage, high spatial resolution, day/night acquisitions, and signal independence of the solar illumination angle. In this study, we obtained coincident optical and SAR images of an agricultural area to investigate the use of SAR imagery for farm management. The optical and SAR data were normalized to indices ranging from 0 to 1 based on the meteorological conditions and sun/sensor geometry for each date to allow temporal analysis. Using optical images to interpret the response of SAR backscatter (σ^o) to soil and plant conditions, we found that SAR σ^o was sensitive to variations in field tillage, surface soil moisture, vegetation density, and plant litter. In an investigation of the relation between SAR σ^o and soil surface roughness, the optical data were used for two purposes: (1) to filter the SAR images to eliminate fields with substantial vegetation cover and/or high surface soil moisture conditions, and (2) to evaluate the results of the investigation. For dry, bare soil fields, there was a significant correlation (r²=.67) between normalized SAR σ^o and near-infrared (NIR) reflectance, due to the sensitivity of both measurements to surface roughness. Recognizing the limitations of optical remote sensing data due to cloud interference and atmospheric attenuation, the findings of this study encourage further studies of SAR imagery for crop and soil assessment.     

Laser-induced fluorescence spectroscopy of dark- and light-adapted bean (Phaseolus vulgaris L.) and wheat (Triticum aestivum L.) plants grown under three irradiance levels and subjected to fluctuating lighting conditions

Fluorescence spectral characteristics associated with growth under different irradiance levels, and during rapidly changing lighting conditions, were measured on healthy bean (Phaseolus vulgaris L.) and wheat (Triticum aestivum L.) plants using a laser-induced fluorescence spectroscopy (LIFS) system. The LIFS system was designed as a prototype of a handheld field remote sensing system and used a tripled Nd:YAG laser to produce ultraviolet (UV) excitation photons at 355 nm. Dark-adapted canopies of the bean and wheat plants grown under 150, 300, or 450 μmol m⁻² s⁻¹ of photosynthetically active radiation (PAR) exhibited LIFS spectra with higher relative fluorescence intensities than emissions from light-adapted plants at all three light levels. Blue/red and blue/far-red leaf fluorescence ratios for both bean and wheat plants increased dramatically as PAR increased, but red/far-red ratios decreased as PAR increased. Light-adapted plants grown under the three light levels were then subjected to several rapidly changing lighting conditions. Plants were exposed sequentially to 150, 300, and 650 μmol m⁻² s⁻¹ PAR from metal halide lamps, followed by a fourth light treatment of 650 μmol m⁻² s⁻¹ PAR from a mixture of metal halide and tungsten-halogen lamps. The tungsten-halogen lamps added significant amounts of near-infrared (NIR) irradiation to the background light environment provided by the metal halide lamps. Results indicated that both bean and wheat canopies generally exhibited stable blue, green, red, and far-red fluorescence emissions when plants were exposed to 150, 300, and 650 μmol m⁻² s⁻¹ PAR from the metal halide lamps. In contrast, when bean and wheat plants were exposed to the NIR-enriched light supplied by the tungsten-halogen lamps, blue and green fluorescence remained stable, but red and far-red fluorescence increased dramatically immediately after exposure to the NIR photons. However, the increased levels of red and far-red fluorescence observed after exposure to NIR light decreased quickly (within 55 s) and returned to “baseline” levels observed at the start of the rapidly changing light experiments. Results indicate that handheld LIFS instruments can be used for remote sensing of plant canopies under a diversity of lighting conditions including full darkness, dawn and dusk lighting environments, and under rapidly changing light environments similar to those encountered on partly cloudy days.     

Steady-state chlorophyll a fluorescence detection from canopy derivative reflectance and double-peak red-edge effects

A series of experiments carried out in a controlled environment facility to induce steady-state chlorophyll a fluorescence variation demonstrate that natural fluorescence emission is observable on the derivative reflectance spectra as a double-peak feature in the 690-710 nm spectral region. This work describes that the unexplained double-peak feature previously seen on canopy derivative reflectance is due entirely to chlorophyll fluorescence (CF) effects, demonstrating the importance of derivative methods for fluorescence detection in vegetation. Measurements were made in a controlled environmental chamber where temperature and humidity were varied through the time course of the experiments in both short- and long-term trials using Acer negundo ssp. californium canopies. Continuous canopy reflectance measurements were made with a spectrometer on healthy and stressed vegetation, along with leaf-level steady-state fluorescence measurements with the PAM-2000 Fluorometer during both temperature-stress induction and recovery stages. In 9-h trials, temperatures were ramped from 10 to 35 °C and relative humidity adjusted from 92% to 42% during stress induction, returning gradually to initial conditions during the recovery stage. Canopy reflectance difference calculations and derivative analysis of reflectance spectra demonstrate that a double-peak feature created between 688, 697 and 710 nm on the derivative reflectance is a function of natural steady-state fluorescence emission, which gradually diminished with induction of maximum stress. Derivative reflectance indices based on this double-peak feature are demonstrated to track natural steady-state fluorescence emission as quantified by two indices, the double-peak index (DPi) and the area of the double peak (A_dp). Results obtained employing these double-peak indices from canopy derivative reflectance suggest a potential for natural steady-state fluorescence detection with hyperspectral data. Short- and long-term stress effects on the observed double-peak derivative indices due to pigment degradation and canopy structure changes were studied, showing that both indices are capable of tracking steady-state fluorescence changes from canopy remote sensing reflectance.     

Imaging chlorophyll fluorescence with an airborne narrow-band multispectral camera for vegetation stress detection

Progress in assessing the feasibility for imaging fluorescence using the O₂-A band with 1 nm full-width half-maximum (FWHM) bands centered at 757.5 and 760.5 nm is reported in this paper. Multispectral airborne data was acquired at 150 m above ground level in the thermal, visible and near infrared regions yielding imagery at 15 cm spatial resolution. Simultaneous field experiments conducted in olive, peach, and orange orchards (water stress trials), and an olive orchard (variety trial) enabled the detected variability in fluorescence emission to be examined as function of stress status. In a parallel modelling activity the coupled leaf-canopy reflectance-fluorescence model, FluorMOD, was used to assess fluorescence retrieval capability by the in-filling method, as well as by fluorescence indices from the published literature. Fluorescence retrievals using the in-filling method, the derivative index D702/D680 and reflectance indices R690/R630, R761-R757, and R761/R757 yielded the best results in the simulation study, while demonstrating insensitivity to leaf area index (LAI) variation. The fluorescence in-filling method, derivative index D702/D680, and R761-R757 were the indices least affected by chlorophyll a + b (Cab) variation. On the other hand, other published indices for fluorescence detection at leaf and canopy levels exhibited high sensitivity to variations in Cab and LAI, and therefore were considered less suitable for in-field fluorescence detection. The fluorescence signal extraction from airborne imagery using the in-filling method was validated through comparisons with field-measured steady-state fluorescence (Fs) using the PAM-2100 and GFS-3000 instruments, confirming simulation predictions. The water stress experiments conducted on olive and peach orchards demonstrated the feasibility of chlorophyll fluorescence (F) extraction at the tree level from the airborne imagery, yielding determination coefficients r² = 0.57 (olive), and r² = 0.54 (peach). Consistent results were obtained between airborne F and ground truth assimilation (A) measured in the olive variety field experiment under no water stress levels, yielding r² = 0.71.     

Detection of fluorescence from a mixed specimen consisting of micro particles attached to different fluorescent substances using a light waveguide incorporated optical TAS

A “ubiquitous human health care system” will require a monolithic optical total analysis system (TAS) consisting of waveguides and microfluidic channels based on a transparent resin chip. Together with the rapid development of the fluorescent marking method, fluorescence analysis by TAS of mixed-microparticle specimen attached to different fluorescent substances will be necessary. Towards realization of this, we here propose a novel method for using a part of the fluorescence acquired by irradiating microparticles with AC-modulated laser power as light dedicated to the discrimination of fluorescent substances. Since the light power for discrimination was extremely weak, we extracted effective signal components using a lock-in detection method. Then, by comparison with the signal of the original fluorescence, we could determine whether the fluorescence signal was from the microparticles attached to the fluorescent substance to be discriminated. Using a mixed specimen composed of microparticle-attached fluorescent substances with emission peaks of 520 nm and 600 nm, we found that 10% of the acquired fluorescence could successfully determine the specified fluorescent substance as a discrimination signal. The peak value of the discrimination signal was approximately double the amplitude of the stationary noise in the discrimination signal.

     

Electrochemical studies of bovine serum albumin immobilization onto the poly-o-phenylenediamine and carbon-coated nickel composite film and its interaction with papaverine

A biosensor based on bovine serum albumin (BSA) and poly-o-phenylenediamine (PoPD)/carbon-coated nickel (C-Ni) nanobiocomposite film modified electrode has been developed to study the interaction of BSA with papaverine (PAP). The well-dispersed C-Ni nanoparticles were dripped onto the glassy carbon electrode (GCE) surface firstly, and PoPD films were subsequently electropolymerized by cyclic voltammetry (CV) to prepare PoPD/C-Ni/GCE. Finally, the BSA was easily immobilized on the PoPD films via electrostatic adsorption. The morphology and the electrochemical properties of the fabricated composite electrodes were examined by scanning electron microscope (SEM) and electrochemical impedance spectroscopy (EIS), respectively. The interaction of PAP with BSA was monitored by differential pulse voltammetry (DPV), using PoPD as the electrochemical indicator. The binding constant (K), obtained by DPV, was 1.7 × 10⁴ L/mol, which was consistent with the fluorescence analysis. This constructed biosensor also exhibited a fine linear correlation with PAP concentration range of 2.5 × 10⁻⁹-4.5 × 10⁻⁵ mol/L and a detection limit of 8.3 × 10⁻¹⁰ mol/L was achieved by DPV.     

Software monitoring with controllable overhead

We introduce the technique of software monitoring with controllable overhead (SMCO), which is based on a novel combination of supervisory control theory of discrete event systems and PID-control theory of discrete time systems. SMCO controls monitoring overhead by temporarily disabling monitoring of selected events for as short a time as possible under the constraint of a user-supplied target overhead o _t. This strategy is optimal in the sense that it allows SMCO to monitor as many events as possible, within the confines of o _t. SMCO is a general monitoring technique that can be applied to any system interface or API. We have applied SMCO to a variety of monitoring problems, including two highlighted in this paper: integer range analysis, which determines upper and lower bounds on integer variable values; and non-accessed period detection, which detects stale or underutilized memory allocations. We benchmarked SMCO extensively, using both CPU- and I/O-intensive workloads, which often exhibited highly bursty behavior. We demonstrate that SMCO successfully controls overhead across a wide range of target overhead levels; its accuracy monotonically increases with the target overhead; and it can be configured to distribute monitoring overhead fairly across multiple instrumentation points.     

A Hybrid Approach to the 3D High Precision Position Measurement and Particle Tracking in Human Cell Nuclei

In this report, we present a detection and tracking system for fluorescent particles inside a cell nucleus. We present the application of this system to a real biological problem where the same human cell nucleus is scanned in three different states: living, fixed and after a treatment used for fluorescence in situ hybridization (FISH). The change in distances between internal sub-chromosomal structures was analyzed.     

Selectivity tune of fluoride ion sensing for phenolic OH-containing BODIPY dyes

A series of BODIPY dyes bearing phenolic OH in the meso-phenyl moiety were designed and synthesized for colorimetric and fluorescent detection of F⁻ ions by way of O-H?F hydrogen bonding interactions. Both the electron-donating group attached on the meso-phenyl skeleton, such as methoxy group and t-butyl group, and the substituting position of the phenolic OH have dramatic influences on the chemical shift of OH, pK_a of OH, and the binding constant of OH to F⁻. The binding selectivity to F⁻ over other anions may be greatly improved by down-regulating the binding constant to F⁻ through structure optimization. As a result, BODIPY 7 can selectively probe F⁻ in both absorption and fluorescence modes in acetonitrile and does not response to other anions including AcO⁻. The observed structure dependence of the binding selectivity may provide guidelines for the development of new F⁻ colorimetric or fluorescent sensors of high selectivity.     

Fluorescence sensing systems: In vivo detection of biophysical variations in field corn due to nitrogen supply

Leaf and canopy fluorescence properties of field corn (Zea mays L.) grown under varying levels of nitrogen (N) fertilization were characterized to provide an improved N sensing capability which may assist growers in site-specific N management decisions. In vivo fluorescence emissions can occur in the wavelength region from 300 to 800 nm and are dependent on the wavelength of illumination. These light emissions have been grouped into five primary bands with maxima most frequently received from corn at 320 nm (UV), 450 nm (blue), 530 nm (green), 685 nm (red), and 740 nm (far-red). Two active fluorescence sensing systems have been custom developed; a leaf level Fluorescence Imaging System (FIS), and a canopy level Laser Induced Fluorescence Imaging System (LIFIS). FIS sequentially acquires high-resolution images of fluorescence emission bands under darkened laboratory conditions, while LIFIS simultaneously acquires four band images of plant canopies ≥1 m² under ambient sunlit conditions. Fluorescence emissions induced by these systems along with additional biophysical measures of crop condition; namely, chlorophyll content, N/C ratio, leaf area index (LAI), and grain yield, exhibited similar curvilinear responses to levels of supplied N. A number of significant linear correlations were found among band emissions and several band ratios versus measures of crop condition. Significant differences were obtained for several fluorescence band ratios with respect to the level of supplied N. Leaf adaxial versus abaxial surface emissions exhibited opposing trends with respect to the level of supplied N. Evidence supports that this confounding effect could be removed in part by the green/blue and green/red ratio images. The FIS and LIFIS active fluorescence sensor systems yielded results which support the underlying hypothesis that leaf and canopy fluorescence emissions are associated with other biophysical attributes of crop growth and this information could potentially assist in the site-specific management of variable-rate N fertilization programs.     

Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device

To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli (E. coli) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard.     

Fluorescence turn-on of easily prepared fluorescein derivatives by zinc cation in water and living cells

By combining a sulfonated β-naphtol as receptor, a hydrophilic fluorescence probe with extreme sensitivity (7 ppb) and selectivity for zinc ion has been designed and synthesized in a simple and efficient way. Based on the inhibition of internal charge transfer (ICT) process, This “turn-on” type fluorescent sensor molecule features high fluorescence increase (13 fold) at 508 nm as well as a red-shift from 371 to 430 nm over most competitive heavy and transition metal (HTM) ions in water (pH 7.4). Furthermore, cell imaging experiments show that this fluorescence probe might be used for monitoring Zn²⁺ within biological samples. The role of this sensor molecule in living cells was further confirmed with the treatment of N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine. And the impact of water-solubility and liposolubility of such fluorescence probe on biological application has been deeply discussed.     

An organically modified sol–gel membrane for detection of lead ion by using 2-hydroxy-1-naphthaldehydene-8-aminoquinoline as fluorescence probe

A fluorescent reagent, 2-hydroxy-1-naphthaldehydene-8-aminoquinoline (HNAAQ) was synthesized, and an organically modified sol–gel membrane for detection of lead ion by using HNAAQ as fluorescence probe was fabricated. Under the optimum conditions, by a coplanar effect and the degree of molecular conjugation due to the complexation of Pb²⁺ with HNAAQ the relative fluorescence intensity I₁₀₀/I₀ of the sensing membrane is linearly increased over the Pb²⁺ concentration range of 1.9 × 10⁻⁷ to 1.9 × 10⁻⁴ mol/L with the detection limit of 8.3 × 10⁻⁸ mol/L. The preparation of this organically modified sol–gel membrane and its characteristics were investigated in detail.