Real Time pH Measurements in the Intact Rat Conceptus Using

Chapter 22

Real Time p H Measurements i n the Intact Rat Conceptus Using Ultramicrofiber-Optic Sensors 1,3

Downloaded by UNIV OF TEXAS AT AUSTIN on August 29, 2017 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/bk-1998-0690.ch022

Weihong Tan

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Bjorn A . Thorsrud , Craig Harris , and Raoul

1,5

Kopelman

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Department of Chemistry, University of Michigan, Ann Arbor, M I 48109-1055 Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, Ann Arbor, M I 48109-2029

Real time measurements of p H have been made i n single, viable, intact rat conceptuses during the period o f organogenesis, using newly developed ultramicrofiberoptic chemical sensors. The fiberoptic sensors are constructed using a novel technology based on photo -nanofabrication o f optical probes and near-field photochemical synthesis. The p H sensors are a thousandfold smaller, with a millionfold reduction i n necessary sample size, and show at least a hundredfold reduction i n response time, when compared to other optical sensors currently available. In the present application, biosensors are inserted into the extraembryonic fluid ( E E F ) compartment o f an intact rat conceptus, which is positioned i n a customized perifusion chamber. Viability is maintained and exposures are controlled during p H determinations, while causing no damage to or leakage of fluid from the surrounding visceral yolk sac ( V Y S ) . Static determinations o f p H i n intact rat conceptuses o f varying gestational ages showed decreasing p H with conceptal age. Dynamic alterations i n p H were also measured in response to several variations i n environmental conditions and specifically during exposure to a cellular thiol oxidant, diamide.

A n understanding o f the functional, dynamic responses o f living cells and complex organisms to endogenous and exogenous signals has long been a goal of biologists from both fundamental and applied disciplines. Many traditional methods for determination o f cellular, biochemical and molecular functions have, o f necessity, been invasive, requiring destruction o f the cell or organism. Micro-optical probes and ultra small microelectrodes have been developed to study minute functional biological units in vivo and in vitro {1-6), including noninvasive monitoring o f biochemical events i n periportal and pericentral hepatocytes i n the intact liver (7,8). Generally, measurements by conventional methods have been determined exogenously to the 3

Current address: Department of Chemistry, University of Florida, Gainesville, FL 32611. Current address: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055. Corresponding author.

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©1998 American Chemical Society Akmal and Usmani; Polymers in Sensors ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


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biological sample(4,5) or following tissue disruption (9,70). One of the limitations of these conventional techniques has been the absence of probes small enough to be used i n extremely small, fragile and dynamic biological systems, such as early embryos and single cells. Irrespective of these specific limitations, the use of fiberoptic chemical biosensors has been growing for both environmental and biological applications (7,2). However, fiberoptic sensors are usually larger than 100 μτη and are thus inappropriate for single cell operations. In addition, response times have been relatively long, typically i n the range of seconds to minutes, making determinations of rapid responses impractical. W e have recently developed ultrasmall optical fiber sensors (11,12) for spatially resolved measurements i n small organisms and single cells. Sensor sizes have been reduced down to 0.1 μπι and have sample volume requirements of only femtoliters. Additionally, these sensors are capable of very rapid (millisecond) monitoring of chemical and biological reactions. Ultrasmall optical fiber sensors are prepared using a photo-nanofabrication technology based on near-field photochemical synthesis and nanofabricated optical probes (11-13), utilizing optical fiber tips (13,14). The working range of optical fiber sensors includes probes with tip dimensions i n the submicrometer to tens of microns range. They are fabricated by incorporating a derivative of fluoresceinamine, N-fluoresceinylacrylamide ( F L A C ) into an acrylamide and Ν,Ν-methylenebis (acrylamide) copolymer that is attached covalently to a silanized fiber tip surface through near-field photopolymerization (77). The insert i n Figure 1 shows a schematic drawing of the fiberoptic p H sensor tip. In order to enhance the working ability of the minkturized fiber optic Isensors, internal calibration methods have been developed (72) and are based on the fluorescence intensity ratios obtained from different wavelengths of the same emission spectrum for a single dye elicited by a single excitation signal. This procedure is highly effective for small-sized sensors, especially when dye species absorption differences are also utilized (72). The first significant biological applications of the ultramicrofiberoptic chemical sensors are demonstrated here using the rat whole embryo culture system. Embryo explantation, growth and culture conditions are previously described (70). Explanted, cultured, viable, gestational day 10 to 12 ( G D 10-12) rat conceptuses (15,16) are carefully placed inside a customized perifusion chamber, which is positioned on the stage of an inverted fluorescence microscope, as shown in Figure 1. This apparatus is able to maintain the viability of G D 10-12 rat conceptuses in serum-free medium for over an hour of monitoring. A schematic of how the miniaturized fiberoptic chemical sensor is inserted and positioned i n the E E F space of the conceptus is shown in Figure 2. The spectroscopic protocol is described in detail (72). A calibration curve was obtained from p H measurements of Hank's Balanced Salt Solution ( H B S S ) . Preliminary experiments, using H B S S , without the embryo present, verified the ability of the submicrometer fiberoptic sensor to discrirninate 0.1 p H unit changes in the p H range of 6.6 to 8.6. Next, G D 10 and 12 conceptuses were analyzed for differences in p H as a function of advancing gestational age. E E F p H measurements, determined by using the fiber optic sensor, show values of 7.50 to 7.56 i n the 10-16 somite, G D 10 rat conceptus and p H values of 7.24 to 7.27 i n the 32-34 somite, G D 12 conceptus (see Table 1). These values are i n good agreement with previous results obtained by using large numbers of homogenized mouse embryos at comparable stages of development, where p H was determined via a radiochemical accumulation method (10). Our new method, i n contrast, uses a single, live conceptus for the p H measurements. The isse of a single embryo has numerous advantages over pooled embryos, i n that it is possible to maintain structural and functional integrity, while monitoring static and dynamic p H changes i n the E E F . The conceptus also serves as its own control, can be accurately characterized as to developmental stage, can be monitored spatially and temporally i n real time and can be returned to whole embryo culture to evaluate other relevant endpoints later in development

Akmal and Usmani; Polymers in Sensors ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


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Microscope Optics

Computer Data Integration

Optical Multichannel Analyzer

Figure 1. Schematic diagram o f the fiberoptic perifusion system, depicting the intact rat conceptus in the customized monitoring chamber, located on the stage of an inverted fluorescence microscope. The emissions signal originating from the tip of the p H sensor inserted into the extraembryonic fluid compartment is detected through the microscope optics, directed to an optical multichannel analyzer ( O M A ) and integrated using a micro computer. The tip of the vjlttarnicrofiberoptic p H sensor has been amplified and is shown schematically in the figure insert



Figure 2. Schematic representation of the rat conceptus with its associated tissues, showing placement of the ultramicrofiberoptic sensor inside the extraembryonic fluid compartment



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The miniaturization of this fiber optical sensor has not occurred at the expense of response times, with current sensors able to respond to events of shorter than 100ms (72). W e made use of this feature i n deteraiining the dynamic response of a rat conceptus to alterations i n its in vitro environment. Experiments were conducted to ascertain the ability of the G D 12 rat conceptus to respond to changes in its environment and to selected chemical agents. Three different experimental protocols were used. First, the perifusate bathing the embryo was saturated with 100% nitrogen, to create a condition of hypoxia. The measured p H over time did n o i vary significantly from the initial control readings. Even though hypoxia does not immediately alter hydrogen ion concentrations in the conceptus as determined by measiiring E E F p H , it does not preclude the possibility that intracellular changes are taking place within cells of the V Y S or embryo proper, but not affecting the equihbrium between E E F and intracellular fluid compartments. Second, conceptal response was determined by monitoring E E F for p H , when the perifusate, containing H B S S , was altered over a p H range of 6.6 to 8.6. A g a i n , the measured p H in E E F did not result in any significant variations from control levels. Finally, the dynamic effects of a thiol oxidant, diamide (used to oxidize intracellular glutathione ( G S H ) to glutathione disulfide ( G S S G ) and induce oxidative stress i n the embryo) was evaluated. A s shown i n Figure 3, a 500 μ Μ solution o f diamide i n the perifusate ( H B S S , p H 7.4) results i n an initial rapid decrease i n p H over the first 30 seconds, followed by a slower downward trend thereafter. A n absolute decrease of about 0.3 p H units occurs within about 3 rninutes. In conclusion, the ultramicrofiberoptic p H sensor is shown to be able to discriminate p H changes of less than a tenth of a p H unit over the range of one p H unit above and below the physiologic p H of 7.4. The p H measurements were obtained in real time, on a single, intact, viable rat conceptus under conditions of environmental change and direct chemical exposure. The insertion of the ultrasmall sensor through the visceral yolk sac into the E E F appeared to cause no damage to or leakage from the involved tissues. This demonstrates the advantage of an essentially non-invasive approach, as compared to conventional means, necessitating the disruption of large numbers of conceptuses to obtain less sensitive and indirect measurements. Conceptuses of ascending developmental age undergo an acidification of the E E F . This finding is consistent with reports using accumulation of C-dimethadione (5,5'dimethyloxazolidine-2,4-dione) or D M O , to determine tissue p H (17,18). Alterations in environmental conditions showed the ability of the rat conceptus to respond to certain external stimuli by maintaining normal intraconceptal p H . O n the other hand, diamide, at a concentration of 500 μΜ, produced a very drastic decrease i n p H over the 30 seconds of chemical exposure. This thiol oxidant may compromise the normal buffering capacity of rat conceptuses through disruption of energy dependent ion compartmentalization or inhibition of H pumps. The observed p H changes (-0.3 p H units) are of a magnitude capable of dramatically altering normal cell function, as has been shown in several other cell systems (79). This observation is important, considering the evidence that changes in intracellular p H may be instrumental in the molecular regulation of differentiation and cell proliferation during development. Intracellular thiol status has also been implicated i n the regulation of the same developmental processes. Biological hydrogen ion pumps are, i n fact, known to be particularly sensitive to disturbances in intracellular thiol status. The lysosomal A T P dependent H + pump is selectively inhibited by the sulfhydryl reagent N ethylmaleimide ( N E M ) , at doses that do not affect other ion pumps (20). The ability of ultramicrofiberoptic sensors to measure p H changes, in real time, i n the intact rat conceptus, demonstrates their potential applications for dynamic analysis in small multicelluar organisms. Working sensor probe dimensions and response characteristics also make this approach feasible for use i n single cells. The application of this novel technology to studies of developmental regulation, pharmacokinetics, 14

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TABLE 1


Measurements of pH in the extraembryonic fluid (EEF) of whole rat conceptuses maintained in vitro as determined using the micro-fiberoptic pH sensor. Day of Gestation

Somite Number

pH

10

10-16

7.63 ±0.07

11

22-26

7.37 ±0.07

12

32-34

7.26 ±0.00

T i m e (minutes)

Figure 3. Representative dynamic p H response during exposure to 500 μ Μ diamide, determined in the extraembryonic fluid of single, viable, G D 12 rat conceptus, using the ultramicrofiberoptic p H sensor. Diamide is added at t=0 and the decrease in p H during the first 30 seconds was determined to be 0 . 1 8 ± 0 . 0 1 p H units (n=3 conceptuses).


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toxicology and physiology w i l l provide valuable spatial and temporal information not heretofore available using conventional techniques. Acknowledgments Supported by N I H grants 1RO1GM50300 and ES05235and by a grant from the Physical Optics Corporation, Torrance, C A .

Literature Cited


1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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Real Time pH Measurements in the Intact Rat Conceptus Using

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