In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Podsumowanie

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

Streszczenie

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO₂, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO₂ and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

Wprowadzenie

A key role of the TME in cancer progression and therapy is increasingly appreciated¹. Among important physiological parameters of the TME in solid tumors, tissue hypoxia², acidosis^3,4, high reducing capacity⁵, elevated concentrations of intracellular GSH^6,7, and interstitial Pi⁸ are well documented. Noninvasive in vivo pO₂, pH, Pi, GSH, and redox assessments provide unique insights into the biological processes in TME, and help advance tools for pre-clinical screening of anti-cancer drugs and TME-targeted therapeutic strategies. A reasonable radiofrequency penetration depth in tissues by magnetic resonance imaging (MRI) and low-field EPR-based techniques makes them the most appropriate approaches for noninvasive assessment of these TME parameters. MRI relies largely on imaging water protons and is widely used in clinical settings to provide anatomical resolution but lacks functional resolution. The phosphorus-31 nuclear magnetic resonance (³¹P-NMR) measurements of extracellular Pi concentration and pH based on a signal from endogenous phosphate are potentially attractive for TME characterization, but are normally masked by several times higher intracellular Pi concentrations^9,10. In contrast to this, EPR measurements rely on spectroscopy and imaging of specially designed paramagnetic probes to provide functional resolution. Note that exogenous EPR probes have an advantage over exogenous NMR probes due to the much higher intrinsic sensitivity of EPR and absence of endogenous background EPR signals. The recent development of a dual function pH and redox nitroxyl probe¹¹ and multifunctional trityl probe¹² provides unsurpassed opportunities for in vivo concurrent measurements of several TME parameters and their correlation analyses independent on probe distribution and time of measurement. To our knowledge, there are no other methods available to concurrently assess in vivo physiologically important chemical TME parameters in living subjects, such as pO₂, pH_e, Pi, redox, and GSH.

Probes for In Vivo Functional Measurements:

Figure 1 shows chemical structures of the paramagnetic probes used to access TME parameters, which include particulate and soluble probes. High functional sensitivity, stability in living tissue, and minimal toxicity are a few benefits that make particulate probes preferred over soluble probes for in vivo EPR oximetry. For example, particulate probes have increased retention times at the site of tissue implant compared to soluble probes allowing for longitudinal measurement of tissue pO₂ over several weeks. On the other hand, soluble probes outperform particulate probes by providing spatial-resolved measurements using EPR-based imaging techniques as well as allowing concomitant analyses from multiple functionalities (pO₂, pH, Pi, redox, and GSH).

Figure 1. Chemical structures of the paramagnetic probes that assemble TME assessment assay. This includes the particulate pO₂ probe, LiNc-BuO (R = -O(CH₂)₃CH₃), and soluble probes: dual function pH and redox probe, NR; GSH-sensitive probe, RSSR; and multifunctional pO₂, pH, and Pi probe of the extracellular microenvironment, the HOPE probe. The synthesis of these probes has been described in the provided references ^11,12. Please click here to view a larger version of this figure.

Protokół

All animal work was performed in accordance with WVU IACUC approved protocol.

1. Probe Synthesis and Calibration

1. Particulate pO₂-sensitive LiNc-BuO probe
   Note: LiNc-BuO microcrystals are synthesized and prepared as described in reference¹³. They are very stable and can be kept at room temperature for years. The EPR linewidth of the LiNc-BuO particulate probe is a pO₂-sensitive parameter. LiNc-BuO microcrystals demonstrate ideal linear dependence of the linewidth on oxygen concentration in the range from anoxic conditions up to 760 mmHg of pO₂ partial pressure¹³, with the values of intrinsic linewidth in the absence of oxygen and the slope of the oxygen dependence (measured in mG/mmHg) slightly varying for different batches of the microcrystal preparation. Therefore, calibration is required for every particular batch.
   1. Weigh 60 mg of LiNc-BuO microcrystals.
   2. For oxygen sensitivity calibration, suspend microcrystals in 3 mL of Dulbecco's Modified Eagle Media (DMEM) medium (in a concentration of 20 mg/mL) and sonicate for 5 min on ice with a probe sonicator at 20 kHz using 7 W power in a 5-mL glass round-bottom tube.
   3. Place 1 mL of the sonicated microcrystals in a glass tube in the surface coil resonator of the L-band (1.2 GHz) EPR spectrometer and acquire continuous waves (CW) EPR spectra at the physiological temperature of 37 °C and oxygen concentrations of 0, 1, 2, 4, 8, and 20.9%. Maintain the oxygen concentration by bubbling the solution with gas mixture delivered from a gas controller and maintain the temperature using a water bath attached to a thermostat. Use the following EPR spectrometer acquisition parameters: modulation amplitude, 100 mG; modulation frequency, 100 kHz; sweep width, 5 G; sweep time, 60 s.
   4. To achieve a better signal-to-noise ratio (SNR), use a modulation amplitude value of 60% of the linewidth (e.g., use a modulation amplitude 0.6 G for the linewidth of 1 G).
   5. Alternative simplified calibration procedure: Record the EPR spectra in air-bubbled and anoxic solutions¹⁴. In the latter case, maintain anoxia in the samples by the addition of 10 mM glucose and 100 U/mL glucose oxidase to 1 mL probe solutions according to reference¹⁴.
   6. Fit the EPR spectra with the Lorentzian function to find the line width, LW. Evaluate the sensitivity of microcrystals to pO₂ as a slope of the dependence of LW on pO₂, namely as a value of (LW_air−LW_anoxia)/pO_2air, where LW_air and LW_anoxia are spectra linewidth in air and anoxia conditions, respectively; pO_2air = 152 mmHg.
2. Dual function pH and redox probe, NR
   Note: The NR probe is synthesized as described in reference¹¹. It is stable at room temperature both as a solid and in aqueous solutions. The synthesized NR probe is kept at 4 °C. The nitrogen hyperfine splitting, a_N, and the signal amplitude decay rate are the spectral parameters of the NR probe that are sensitive to pH (probe pK_a = 6.6 at 37 °C, range of pH sensitivity from 5.6 to 7.6) and to the reducing capacity of the probe microenvironment, respectively.
   1. Remove the NR from the freezer and allow the container to warm to room temperature (10 - 15 min). Weigh out 6.34 mg of the NR, dissolve it in 1 mL of saline solution, and adjust the pH to 7.2 with small aliquots of HCl or NaOH using a pH meter. Use the prepared NR solution (10 mM) as a stock solution.
   2. Perform the pH calibration of the NR probe as follows (see reference¹¹). First, add 0.1 mL of the NR stock solution to 0.9 mL of 2 mM Na-phosphate buffer, 150 mM NaCl. Titrate the obtained 1 mM NR solution with aliquots of HCl or NaOH to the required pH using a pH meter. Control the temperature using a water bath attached to a thermostat.
   3. Record the EPR spectra of the samples in 1.5-mL microcentrifuge tubes using the L-band EPR spectrometer. Use the following EPR spectrometer acquisition parameters: modulation amplitude, 2.5 G; modulation frequency, 100 kHz; sweep width, 60 G; sweep time, 20 s.
   4. Measure the hyperfine splitting constant (a_N) as half of the distance between low- and high-field components of the EPR spectra and plot it versus pH, to provide the calibration curve for L-band EPR measurements of pH.
3. GSH-sensitive RSSR probe
   Note: The RSSR probe is synthesized as described in reference¹⁵. Store the synthesized NR probe at 4 °C. The lipophilic RSSR disulfide biradical compound easily diffuses across the cell membrane to react with intracellular GSH and provide a reliable approach for determining GSH in vivo using EPR^16,17. This method is based on the high reaction rates of the predominant intracellular pool of GSH thiols with the RSSR probe. The reaction of the RSSR biradical with GSH splits its disulfide bond (see Scheme 1) resulting in the cancellation of spin exchange between the two radical fragments and manifesting in a decrease of the biradical spectral components and corresponding increase of the monoradical components. For the biradical RSSR probe, the rate of the increase of the amplitude of the monoradical component is proportional to the GSH concentration and is a convenient GSH-sensitive EPR spectral parameter. To evaluate GSH concentration from the in vivo EPR measurements, the preceding calibration of the rate of the RSSR reaction with GSH at the corresponding temperature and pH has to be performed as follows.
   1. Remove the RSSR from the freezer and allow the container to warm to room temperature (10-15 min). Weigh out 4.05 mg of the NR and dissolve it in 1 mL of DMSO solution. Use the prepared RSSR solution (10 mM) as a stock solution.
   2. Determine the value of the rate constant, k_obs, of the reaction of RSSR with GSH at the desirable temperature and pH as follows.
   3. First, add 20 µL of the RSSR stock solution (10 mM) to 0.98 mL of 1 mM Na-phosphate buffer, pH 7.2, 150 mM NaCl, to obtain a 0.2 mM RSSR probe solution.
   4. Prepare solutions of 1, 2, and 5 mM concentrations of GSH in 0.1 M Na-phosphate buffer at pH 7.2. To accurately evaluate GSH concentration in the cells of the targeted organ from the in vivo measurements, the in vitro calibration has to be performed at a pH close to the value of the intracellular pH.
   5. Mix equal volumes of 0.2 mM RSSR solution and one of the GSH solutions prepared in step 1.3.4. for a final concentration of the probe at 0.1 mM and of GSH at 0.5, 1, or 2.5 mM.
   6. Immediately after RSSR and GSH solution mixing, place the sample in the EPR resonator and record the EPR spectra every 12 seconds for 10 minutes. Then calculate the kinetics of the increase of the monoradical spectral amplitude. Use the following EPR spectrometer acquisition parameters: modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 60 G; sweep time, 10-60 s.
   7. Fit the measured EPR kinetics to the monoexponents and calculate the time constant of the exponential kinetics, τ. The linear regression (1/τ = k_obs × [GSH]) provides the observed rate constant value of the reaction between GSH and RSSR (e.g., at 34 °C and pH 7.2, k_obs = 2.8 ± 0.2 M^-1s^-1)¹¹.
4. Multifunctional HOPE probe for pO₂, pH, and Pi assessment
   Note: The monophosphonated trityl HOPE probe is synthesized as described in reference¹² and is kept at 4 °C. The CW EPR spectra of HOPE at pH << pK_a (A - acid form) and pH >> pK_a (B - basic form) are represented by the doublets due to phosphorus hyperfine splitting, a_P. The typical instrument settings are as follows: modulation amplitude, 37.5 mG; modulation frequency, 100 kHz; sweep width, 0.9 G; sweep time, 20-60 s. At intermediate pH (5 < pH < 8) the EPR spectrum of HOPE probe is characterized by a quartet when both A and B states are present. The individual EPR linewidth of the HOPE is a pO₂ marker (accuracy, ≈ 1 mmHg; pO₂ range, 1-100 mmHg). The fraction of protonated HOPE (A form) is a pH marker in the range from 6 to 8.0 (accuracy, ± 0.05). The value of the proton exchange rate (expressed in mG) with Pi extracted by spectra simulation is a Pi marker (accuracy, ± 0.1 mM, range, 0.1-20 mM). Calibration procedures are performed at physiological temperature (37 °C), solution ionic strength (NaCl, 150 mM), and HOPE probe concentration of 0.2 mM, as previously described in references^12,18, and detailed below.
   1. Remove the HOPE probe from the freezer and allow the container to warm to room temperature (10-15 min).
   2. Weigh out 10.7 mg of the HOPE probe, dissolve it in 1 mL of saline solution, and adjust the pH to 7.4. Add 20 µL of the prepared stock solution of the HOPE (10 mM) to 0.98 mL of the saline solution to obtain a 0.2 mM HOPE probe solution.
   3. For probe calibration of the pH, titrate 0.2 mM of the HOPE probe solution by the addition of a small volume of NaOH or HCl, with the final dilution of sample less than 1%. Measure the pH with a pH electrode calibrated at 37 °C using the pH values for the reference solution recommended by National Bureau of Standards (U.S.). Use a jacketed reaction beaker attached to a circulator to carefully maintain temperature of the reference and titrated solutions during pH measurements. Maintain anoxic conditions by the addition of 10 mM glucose and 100 U/mL glucose oxidase to the probe solutions.
   4. Acquire the EPR spectra of the A and B forms at pH ≤ 5 and pH ≥ 8, respectively, in anoxic conditions in the absence of phosphate.
   5. Use the corresponding spectra to obtain intrinsic spectral parameters. Namely, simulate the spectral line as the convolution of the Lorentzian function with the Gaussian function that approximates the unresolved super hyperfine structure of the HOPE probe. The fitting of the calculated EPR spectra to the experimental spectra yields the values of a_P and Lorentzian linewidth (ΔL_pp), determined by the transverse relaxation rate, 1/T₂ (where 1/T₂ = (√3/2)L_pp for the measured derivative of the RF absorption line in CW EPR), and the linewidth of Gaussian distribution, G.
      Note: The following are the parameters obtained from the spectra measured at the conditions specified in step 1.4.2: a_P(A) = 3.63 G, a_P(B) = 3.37 G; 1/T₂(A) = 23.6 mG; 1/T₂(B) = 9 mG; G(A) = 40 mG; G(B) = 45 mG (see reference⁸).
   6. Acquire the EPR spectra of the HOPE at intermediate pH (5 < pH < 8). Simulate the high-filed component of the acquired EPR spectra using the theory of exchange between several sites in non-coupled or loosely coupled systems adapted from reference¹⁹ as previously described¹⁸. Use the intrinsic parameters obtained for A and B states (see step 1.4.5) to decrease the number of variables. Fit the calculated spectra to the experimental ones to find the values of the fraction A (p_A), and plot the dependence of the p_A value on pH. Use the dependence of p_A on pH in further studies as a pH calibration curve.
      Note: Fitting the pH dependence of p_A with a standard titration curve provides the value of the dissociation constant, pK_a (HOPE) = 6.9⁸. In in vivo studies, acquiring the full EPR spectra of the HOPE probe is impractical because of the additional time needed to acquire the gap between low- and high-field components of the phosphorus hyperfine splitting in the EPR spectrum. Therefore, in further exemplified studies only the high-filed component of the EPR spectrum has been measured and analyzed.
   7. For probe calibration of pO₂, acquire EPR spectra of the HOPE probe at various oxygen concentrations.
   8. Control the pO₂ values of the solutions by bubbling with gas mixture delivered from a gas controller. Control the solution temperature (37 °C) using a water bath attached to a thermostat.
   9. Simulate the EPR spectra and fit them to the experimental ones as described in step 1.4.6 to determine the values of oxygen-induced relaxation rates.
      Note: The values of oxygen-induced relaxation rates were 0.49 mG/mmHg both for A and B forms of the HOPE probe as measured at 37 °C⁸.
   10. For probe calibration of [Pi], acquire EPR spectra of the HOPE probe at various phosphate concentrations. Use the HOPE radical solution with a pH value near pK_a (pK_a = 6.9 at 37 °C)¹⁸ and titrate it with various phosphate concentrations. Maintain the temperature and gas composition as described in above steps.
   11. Simulate the EPR spectra and fit them to the experimental ones as described in step 1.4.6 to determine the values of Pi-induced exchange rate.
       Note: The dependence of the Pi-induced exchange rate on [Pi] is used as calibration in further studies.

2. Mouse Models of Breast Cancer

1. MMTV-PyMT spontaneous tumor model
   1. Use 4-8 week-old female Friend virus B-type susceptibility/NIH (FVB/N) mouse mammary tumor virus promoter (MMTV) polyoma middle-T antigen (PyMT+) mice with spontaneously formed mammary tumors for in vivo EPR studies.
   2. For comparison of tissue microenvironments of normal mammary glands and tumors, use age-matched littermate females deficient in the PyMT oncogene (PyMT−, "wild type")²⁰.
   3. Subject the mice to L-band EPR spectroscopy once per week for four weeks during isoflurane anesthesia (see probe delivery below).
   4. Anesthetize the mouse using an air-isoflurane mixture (3% isoflurane), and place the mouse on an adjustable table in a right, lateral position with the tumor (mammary glands) close to the surface coil resonator.
   5. After mouse placement, administer the probe by intratissual (i.t.) injection, tune the EPR spectrometer, and acquire the EPR spectra for 5-10 min.
   6. Measure 2-3 mammary tumors (from MMTV-PyMT+ mice) or non-tumor bearing mammary glands (from PyMT− mice) during the same EPR session.
2. Orthotopic MET-1 tumor model
   1. Grow FVB/N background Met-1 murine breast cancer cells at 37 °C, 5% CO₂, and 95% relative humidity in DMEM containing 10% fetal bovine serum (FBS), 10 µg/mL insulin, 5 ng/mL rhEGF, and 1% PSA (penicillin G sodium, streptomycin sulfate, and amphotericin B) to ~ 80% confluence in a T175 flask.
   2. Aspirate the media and rinse the adherent cells with 10 mL PBS (1.54 mM KH₂PO₄, 155 mM NaCl, and 2.71 mM Na₂HPO₄-7H₂O without calcium chloride or magnesium chloride, pH = 7.4).
   3. Detach the cells by adding 5 mL of 0.25% Trypsin-EDTA solution and rocking the flask. When the cells are detached, add 10 mL DMEM containing 10% FBS to the flask and collect the cells.
   4. Centrifuge the cell suspension at 132 x g for 10 min at 4 °C. Count the cells using a hemocytometer and resuspend to 1 x 10⁶ cells per 100 µL minimal DMEM.
   5. Using an insulin syringe (29G1/2 needle), slowly inject 100 µL of tumor cell suspension into the number 4 mammary fat pads of 8-week-old female FVB/N wild type mice.
   6. Monitor tumor initiation by palpation (appear after approximately 2-3 weeks), growth (visual), and mouse heath (visual) every other day.
   7. Measure tumor dimensions once per week using calipers and determine tumor volumes using the equation:
      

3. Probe Delivery for In Vivo Functional Measurements

1. Use particulate LiNc-BuO probe (Protocol I) for pO₂ measurements in orthotopic tumor models by implanting tumor cells with internalized LiNc-BuO microcrystals as previously described^14,21 and detailed below.
   1. In the case of the MET-1 tumor model, for internalization of LiNc-BuO microcrystals into MET-1 cells, suspend the LiNc-BuO microcrystals in DMEM at a concentration of 20 mg/mL and sonicate with a probe sonicator at 20 kHz using 7 W power in a 5-mL glass round-bottom tube for 5 min on ice.
   2. Add 100 µL (2 mg of LiNc-BuO) of the suspension to a T75 flask with 10 mL culture media containing MET-1 cells (approximately 30% confluent). All procedures take place in a biosafety cabinet and the media contains penicillin and streptomycin to minimize potential infection.
   3. Incubate the cells at 37 °C for 72 h or until they reach ~ 80% confluency.
   4. Aspirate the media. Wash the cells five times with 10 mL PBS. Detach the cells with 5 mL trypsin-EDTA. Collect the cells. Centrifuge as described above in step 2.2.4. Stain a sample of cells with exclusion dye to determine cell viability and quantity.
   5. Suspend the cells at a concentration of 1 x 10⁶ per 100 µL minimal DMEM.
   6. Using an insulin syringe, slowly inject 100 µL of cell suspension containing the internalized LiNc-BuO microcrystals into the number 4 mammary fat pads of 8-week-old female FVB/N wild type mice as described in step 2.2.5.
   7. Monitor tumor initiation and growth as described in steps 2.2.6 and 2.2.7.
2. Use particulate LiNc-BuO probe (Protocol II) in either spontaneous or orthotopic models. Inject LiNc-OBu microcrystals at the site of interest, e.g., into normal mammary glands or mammary tumors, using an insulin syringe.
3. Soluble probes
   1. Anesthetize the mice by inhalation of an air-isoflurane mixture (1.0 L/min delivery and 2-3% of isoflurane) using an anesthesia machine and place them into the gap of the EPR spectrometer.
   2. Tune the instrument, then inject the NR (10-30 µL, 10 mM), HOPE probe (10−30 µL, 0.5-2 mM) in saline, pH 7.2, or RSSR probe in DMSO (10 µL, 10 mM) solutions (i.t.).

4. In Vivo Functional Measurements

1. For EPR spectroscopic measurements, anesthetize the mice by inhalation of air-isoflurane mixture using an anesthesia machine as described in step 3.3.1.
2. Perform functional measurements using L-band (1.2 GHz) EPR spectrometer as follows.
   1. Place the surface coil resonator onto a normal mammary gland or a mammary tumor and tune the spectrometer.
   2. Acquire the EPR spectra from the implanted particulate probe for 5−10 min over several weeks after implantation. In the case of soluble probes, acquire the EPR spectra immediately after probe injection for 5-10 min.
   3. Analyze the EPR spectra of the NR probe to find the hyperfine splitting, a_N, and signal amplitude, I(t). Convert the value of a_N to the pH value using the calibration curve obtained in step 1.2.4. Analyze the rate of decay of the signal amplitude I(t) as relative change from the initial amplitude, I(t = 0), calculated in arbitrary units per second (s^-1).
   4. Fit the increase of the monoradical component of the EPR spectrum of GSH-sensitive RSSR probe to the monoexponents to obtain the time constant of the exponential kinetics to calculate GSH concentration.
   5. Fit the EPR spectra of high-field component of the multifunctional HOPE probe to the experimental ones as described in (step 1.4.5) to yield the values of pH, pO₂ and Pi.

5. Statistical Analysis

1. Perform data processing and statistical analyses. Use a Pearson's r Correlation test (for normally distributed datasets) and Spearman's Rank Order Correlation (for datasets with rejected normality of data distribution) for correlation analyses.

Wyniki

Tissue pO₂ Assessment Using the LiNc-BuO Probes:

Using the procedure described under step 1.1, we performed the calibration of freshly prepared LiNc-BuO microcrystals suspension. Figure 2 shows the typical oxygen dependence of the linewidth of the LiNc-BuO probe, as well as its exemplified EPR sp...

Dyskusje

The presented methods allow for noninvasive in vivo assessment of the critical parameters of the chemical TME, namely pO₂, pH, redox status, and concentrations of interstitial Pi and intracellular GSH. Magnetic resonance techniques, such as MRI and low-field EPR, are the methods of choice for noninvasive in vivo profiling of these TME parameters. MRI visualizes anatomical structures but lacks functional sensitivity. In contrast to MRI, EPR techniques provide functional sensitivity to...

Materiały

Name	Company	Catalog Number	Comments
L-band EPR spectrometer	Magnettech, Germany		L-band (1.2 GHz) electron paramagnetic resonance (EPR) spectrometer for collection in vitro and in vivo spectra of paramagnetic molecules
Temperature & Gas Controller	Noxygen, Germany		Temperature & Gas Controller designed to control and adjust the temperature and gas composition
Sonicator	Fisher Scientific
GSH (L-Glutathione reduced)	Sigma-Aldrich	G4251
MMTV-PyMT  mice	In house
DMEM	Thermo Fisher Scientific	11995065
Met-1 murine breast cancer cells	In house
C57Bl/6 wild type mice	Jackson Laboratory
Trypsin	Thermo Fisher Scientific	25200056
Trypan Blue Exclusion Dye	Thermo Fisher Scientific	T10282
Ohmeda Fluotec 3
Isoflurane (IsoFlo)	Abbott Laboratories
Sodium phosphate dibasic	Sigma-Aldrich	S9763
Sodium phosphate monobasic	sigma-Aldrich	S07051
Sodium Chloride	sigma-Aldrich	S7653
Hydrochloric acid	sigma-Aldrich	320331
Sodium Hydroxide	sigma-Aldrich	S8045
Glucose	sigma-Aldrich
Glucose oxydase	sigma-Aldrich
Lauda Circulator E100	Lauda-Brikmann
pH meter Orion	Thermo Scientific
LiNc-BuO probe	In house		The Octa-n-Butoxy-Naphthalocyanine probe was synthesizided according to ref 13
NR probe	In house		The Nitroxide probe was synthesizided according to ref 11
RSSR probe	In house		The di-Nitroxide probe was synthesizided according to ref 15
HOPE probe	In house		The monophoshonated Triarylmethyl probe was synthesizided according to ref 12