Hui-Hui Zeng

Zinc biosensing with multiphoton excitation using carbonic anhydrase and improved fluorophores

Previously, we had shown that the zinc-dependent binding of certain fluorescent aryl sulfonamide inhibitors could be used with apocarbonic anhydrase II to transduce the level of free zinc as a change in the fluorescence of the inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made possible quantitation of free zinc in the picomolar range with high selectivity, they have only modest absorbance which limits their utility. We describe here the synthesis and properties of two new probes, Dapoxyl sulfonamide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibits a dramatic increase and blue shift in its emission upon binding to holocarbonic anhydrase II, as well as a 20-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anisotropy of BTCS increases fivefold upon binding to the holoprotein, making this probe well suited for anisotropy-based determination of zinc. BTCS and ABD-N are efficiently excited with two photon excitation using 1.5 ps pulses from a titanium sapphire laser, and exhibit the increased zinc-dependent anisotropy response anticipated on the basis of photoselection.

In situ Measurement of Free Zinc in an Ischemia Model and Cell Culture Using a Ratiometric Fluorescence-Based Biosensor

Zinc ion is of growing interest in medicine and biology generally, and especially in the ischemic brain and other tissues. We have developed ratiometric fluorescence-based biosensors for the study of zinc in these systems; the biosensors use apocarbonic anhydrase variants as recognition elements that offer high sensitivity and selectivity. We report continuous in situ, in vivo measurement of nanomolar extracellular zinc in the brain of an animal model of ischemia using a ratiometric fiber optic biosensor. We also report the development of an expressible excitation ratiometric indicator of zinc ion suitable for use in cells that exhibits picomolar sensitivity. Finally, we also report the discovery that the Zn complex of the chelator TPEN seems to be comparably apoptogenic to the free chelator itself.

Instrumentation for fluorescence-based fiber optic biosensors.

This chapter summarizes the construction principles, operation, and calibration of (single-fiber) fluorescence-based fiber optic sensors. These sensors transduce recognition of a chemical analyte by a transducer such as a protein molecule as a change in fluorescence wavelength or lifetime that can be measured remotely through a length of fiber optic. Examples are given of determination of metal ions in aqueous solution by fluorescence ratio and lifetime. Included are descriptions of instruments, alignment procedures, identification of noise sources, use of calibration standards, factors in the use of long fibers for sensing, issues in field and shipboard operation, and probe preparation.

In vivo and intracellular sensing and imaging of free zinc ion

We describe new methods for the study of zinc in biological specimens. Intracellular free zinc was determined at levels down to picomolar using an excitation ratiometric fluorescence-based biosensing approach using a carbonic anhydrase variant as transducer. A new fiber optic sensor suitable for in vivo use is also described using laser excitation and an emission ratiometric approach; the zinc concentration range of sensor response can be selected to fit the application.

Issues in enzyme-based metal ion biosensing in complex media

In developing fluorescence-based biosensors for rapidly determining metal ions such as zinc or copper in complex media such as sea water, serum, cerebrospinal fluid, or the interior of a cell, several issues must be considered. Among these are the selectivity, sensitivity, ease of calibration, speed of response, reversibility, stability, and the ease of immobilization onto a solid substrate. While the first three have been dealt with in the design of our transducer molecule, apocarbonic anhydrase II, the others remain to be considered. In this paper we examine the stability of the apoprotein to storage at various temperatures and pHs with a view to establishing storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a fluorescent-labeled apo-CA variant on quartz to determine its sped, sensitivity, and kinetics of response.

Improved fluorophores for zinc biosensing using carbonic anhydrase

Previously, we had shown that the zinc-dependent binding of certain fluorescent aryl sulfonamide inhibitors could be used with apo-carbonic anhydrase II to transduce the level of free zinc as a change in the fluorescence of the inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made possible quantitation of free zinc in the picomolar range with high selectivity, they have only modest absorbance which limits their utility. We describe here the synthesis and properties of two new probes, Dapoxyl sulfonamide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibits a dramatic increase and blue shift in its emission upon binding to holo-carbonic anhydrase II, as well as a twenty-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anisotropy of BTCS increases five-fold binding to the holoprotein making this probe well suited for anisotropy-based determination of zinc.

Real-time in-situ determination of free Cu(II) at picomolar levels in sea water using a fluorescence lifetime-based fiber optic biosensor

A continuing issue in chemical oceanography and environmental monitoring is the need for frequent and continuous monitoring of analytes in complex matrices such as sea water and ground waters. Particularly for analytes at trace levels such as Cu(II) in sea water, sampling and analysis of discrete specimens is costly, slow, labor intensive, employs ship time inefficiently, and risks error by contamination. We have developed a fluorescence lifetime- based fiber optic biosensor which demonstrates real time determination of free Cu(II) in coastal waters, in situ, with a subpicomolar detection limit.

The effects of zinc supplementation on primary human retinal pigment epithelium

Population-based and interventional studies have shown that elevated zinc levels can reduce the progression to advanced age-related macular degeneration. The objective of this study was to assess whether elevated extracellular zinc has a direct effect on retinal pigment epithelial cells (RPE), by examining the phenotype and molecular characteristics of increased extracellular zinc on human primary RPE cells. Monolayers of human foetal primary RPE cells were grown on culture inserts and maintained in medium supplemented with increasing total concentrations of zinc (0, 75, 100, 125 and 150 μM) for up to 4 weeks. Changes in cell viability and differentiation as well as expression and secretion of proteins were investigated. RPE cells developed a confluent monolayer with cobblestone morphology and transepithelial resistance (TER) >200 Ω*cm2 within 4 weeks. There was a zinc concentration-dependent increase in TER and pigmentation, with the largest effects being achieved by the addition of 125 μM zinc to the culture medium, corresponding to 3.4 nM available (free) zinc levels. The cells responded to addition of zinc by significantly increasing the expression of Retinoid Isomerohydrolase (RPE65) gene; cell pigmentation; Premelanosome Protein (PMEL17) immunoreactivity; and secretion of proteins including Apolipoprotein E (APOE), Complement Factor H (CFH), and High-Temperature Requirement A Serine Peptidase 1 (HTRA1) without an effect on cell viability. This study shows that elevated extracellular zinc levels have a significant and direct effect on differentiation and function of the RPE cells in culture, which may explain, at least in part, the positive effects seen in clinical settings. The results also highlight that determining and controlling of available, as opposed to total added, zinc will be essential to be able to compare results obtained in different laboratories.

Towards early detection of age-related macular degeneration with tetracyclines and FLIM

Recently, we discovered microscopic spherules of hydroxyapatite (HAP) in aged human sub-retinal pigment epithelial (sub-RPE) deposits in the retinas of aged humans (PMID: 25605911), and developed evidence that the spherules may act to nucleate the growth of sub-RPE deposits such as drusen. Drusen are clinical hallmarks of age-related macular degeneration (AMD). We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, also stained the HAP spherules, but in general the HAP-bound fluorescence excitation and emission spectra overlapped with the well-known autofluorescence of the RPE overlying drusen, making them difficult to resolve. However, we also found that certain tetracyclines exhibited substantial increases in fluorescence lifetime upon binding to HAP, and moreover these lifetimes were substantially greater than those previously observed (Dysli, et al., 2014) for autofluorescence in the human retina in vivo. Thus we were able to image the HAP spherules by fluorescence lifetime imaging microscopy (FLIM) in cadaveric retinas of aged humans. These findings suggest that FLIM imaging of tetracycline binding to HAP could become a diagnostic tool for the development and progression of AMD.

Flim of Physiological Free CU(II) in Living Cells with a Protein-Based Biosensor

Recently, the biology of copper ion has excited substantial interest not only in the biomedical field but in oceanography and the environment as well. Rae, et al., famously predicted (Science 284, 805 (1999))that ordinarily no free copper ion would be found in resting cells based on the femtomolar affinity of a yeast copper chaperone. We have developed a biosensor that employs variants of human carbonic anhydrase II (CA II) with very high affinity and selectivity to quantitate free Cu(II) by changes in fluorescence lifetime. Using a membrane-penetrant CA II variant with much higher selectivity for Cu(II) over Zn (II) than wild type, we measured free Cu(II) levels by confocal fluorescence lifetime imaging microscopy in resting PC-12 cells in vitro. We found that the indicator system could be successfully calibrated in the cell, and that the resting level was very low, near the detection limit of ∼10 femtomolar. The implications of the findings are discussed. The authors thank NIH for support (RO1- EB003924-05 [RBT, CAF, HHZ, and BJM] and RC1-GM091081-01 [JRL, HS, and RBT]) and ISS (Champaign, IL) for the use of the Alba confocal FLIM.View Large Image | View Hi-Res Image | Download PowerPoint Slide