A fluorescent chemosensor for calcium with excellent storage stability in water

a n a l y t i c a c h i m i c a a c t a 6 1 1 ( 2 0 0 8 ) 197–204

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A fluorescent chemosensor for calcium with excellent storage stability in water Huarui He ∗ , Kenneth Jenkins, Chao Lin OPTI Medical Systems, Inc., 235 Hembree Park Drive, Roswell, GA 30076, USA

a r t i c l e

i n f o

a b s t r a c t

Article history:

This article describes the design, synthesis and characterization of an optical sensor suitable

Received 22 October 2007

for practical measurement of ionized calcium in serum and whole blood samples. The key

Received in revised form

to the development of this sensor is the identification of a chemically very stable, nitrogen-

19 January 2008

containing, calcium selective ionophore, coupled with a fluorophore having the correct

Accepted 22 January 2008

spectral and electron accepting properties. The slope of the sensor is about 34%/mM in the

Published on line 2 February 2008

typical clinically significant range of 0.32–2.2 mM. This sensor has been implemented into the disposable cartridge, used for commercially available OPTI CCA analyzer with precision

Keywords:

better than ±0.02 mM (1 S.D.). The sensor displays excellent stability against hydrolysis and

Fluoroionophore

oxidation, leading to less than 0.02 mM measurement error after 9 months of wet storage at

Optical sensor

room temperature, up to 30 ◦ C.

Calcium indicator

1.

Introduction

The measurement of ionized calcium in blood or serum is of importance in clinical diagnosis for many diseases, such as hypoparathyroidism, tumor metastasis, renal failure, etc. Traditionally, it was determined in plasma or serum using ion-selective electrodes [1]. However, with the rapid growth of near-patient devices used at the hospital bedside, there is increasing demand for portable systems utilizing small disposable sensors capable of whole-blood measurements. Consequently, the development of practical and inexpensive optical sensors and systems for the clinical determination of ionized calcium in whole blood remains an important area of research [2,3]. There are many optical sensing schemes for calcium involving multiple types of molecules, which have been described, such as ion-exchange between the measured ion and a proton measured with a lipophilic pH-sensitive indicator dye [4,5,34], or interaction of a potential-sensitive dye with a neutral ion carrier [6,7], and fluorescence calcium indicators [8]. Most analytical methods based on neutral ion

∗

Corresponding author. Tel.: +1 770 510 4444; fax: +1 770 510 4445. E-mail address: [email protected] (H. He). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.01.059

© 2008 Elsevier B.V. All rights reserved.

carriers suffer from inherent pH-dependencies (if based on pH indicators or proton exchange), and/or instabilities associated with the leaching of critical components (if based on an ionophore to extract the desired cation into a lipophilic polymer membrane), unless they are plasticizer-free and their components are covalently immobilized [34]. Therefore, fluorescence calcium indicators with a covalently immobilizable group become one of the best choices for a practical useful candidate for such an application. The fluorescence calcium indicator for intra-cellular calcium was first reported by R. Tsien [8] and has drawn a lot of attention since then. A great number of publications have appeared after the following two decades [9–14]. Despite of the difference of fluorophores, almost all of them are based on BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N N tetraacetic acid) aromatized from EGTA [ethylenene glycol bis(␤-aminoethyl)-N,N,N N -tetraacetic acid] with a dissociation constant in the range of micro-molar (see Fig. 1A). For determination of extra-cellular ionized calcium, whose concentration lies in milli-molar range, none of these fluorescent

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Fig. 1 – Structures of calcium ionophores.

calcium indicators is suitable for this application, because their binding to calcium is about one thousand times too tight. One way to weaken the binding strength is to put an electronwithdrawing group such as nitro or halogens on one of the aromatic rings [15,16] (Fig. 1B). Those electron-withdrawing groups do suppress the bindings, but also create some other problems such as fluorescence quenching and synthetic difficulties. Another way to weaken the binding is to use only half of the binding unit of BAPTA, namely o-anisidine-N,N-diacetic

acid (Fig. 1C), which we believe was first reported by Irvine and Da Silva [17], and gave a binding strength in the milli-molar range with adequate selectivity against magnesium in extracellular application. However, the ionophore decomposed in the aqueous solution at pH 7.40 after 1 month of storage at room temperature [18]. A similar instability of BAPTA was also reported, especially for acidic form of BAPTA [19]. We found that the decomposition resulted from de-alkylation of acetic acid from aniline [18]. The instability of this type of ionophore

Fig. 2 – Synthesis of fluoroionophore 8.

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with an acetic acid linked directly to the aromatic nitrogen precluded their application to our system, in which the wet storage stability is an essential requirement. We now report a new and practical fluorescent indicator designed and commercially proven (within the OPTI CCA) for direct determination of ionized calcium at extra-cellular concentrations found in whole blood, serum, or plasma. The respective sensor is constructed by covalent immobilization of a new fluoroionophore (8 in Fig. 2) onto an aminofunctionalized cellulose embedded in a hydrophilic polymer matrix. The OPTI CCA is a portable blood/serum analyzer using optical sensor technologies [33]. The instrument uses blue-LED as an excitation source and photodiode as an emission detector. The measuring parameters include pH, CO2 , O2 , sodium, potassium, calcium, chloride, glucose, urea, total hemoglobin and oxygen saturation.

2.

Experimental

2.1.

Reagents

Solvents and reagents used in the synthesis of fluoroionophore 8 were purchased from Aldrich (Milwaukee, WI) and used without further purification. Analytical grade buffer and inorganic salts were purchased from either Fluka AG (Buchs, Switzerland) or Sigma Co. (St. Louis, MO). The hydrophilic hydrogel D4 was purchased from CadioTech Interantional (Woburn, Massachusetts), and the polyester sheets (MELINEX) from Polymer Supplier (Atlanta, GA).

2.2.

Instruments

Absorbance measurements were performed with a Shimadzu UV2101PC spectrophotometer. Titration of ionophore E was carried out in the following manner. A stock solution of ionophore in methanol was diluted with N(2-hydroxyethyl)piperazine-N -ethanesulfonic acid (HEPES) buffer containing 145 mM NaCl, 4.5 mM KCl in a volumetric flask, the required amount of solid calcium chloride was added and the solution’s absorption spectrum measured. Fluorescence measurements were performed with an ISS PC1 photon counting fluorometer with a xenon lamp as excitation source. A sensor disk (25 mm, see below) was placed into a custom flow-through cell fitted into the fluorometer. HEPES buffer solutions of the appropriate pH and ion concentrations were then pumped through the cell prior to collection of emission and excitation spectra. Buffer pH values were measured at room temperature (∼22 ◦ C) and 37 ◦ C with a Corning pH meter 125 and AVL-Roche 987 analyzer, respectively. All NMR data were collected at room temperature by QE 300 (Nicolet/GE), 300 mHz, with 0.1% TMS as standard. Elemental analyses were performed by Galbraith Laboratories Inc., Knoxville, TN.

2.3. Immobilization of calcium fluoroionophore (8) onto aminocellulose Aminocellulose (5 g) [20] was suspended in 50 mL 2.5% aqueous sodium carbonate for 30 min, filtered, re-suspended in 50 mL DMF for 30 min, filtered, washed twice more with DMF

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in order to replace trapped water. The washed cellulose was then transferred into a flask containing 8 (0.25 g, 0.3 mmol), N,N-dicyclohexyl-1,3-carbodiimide (DCC, 0.62 g, 3 mmol) and N-hydroxysuccinimide (NHS, 0.35 g, 3 mmol) in anhydrous DMF (20 mL) and the suspension was stirred at room temperature for 20 h. The yellow cellulose fiber was filtered, washed with DMF (5× 50 mL), water (50 mL), 0.2N HCl (2× 50 mL), water (50 mL), 1.0N NaOH (2× 50 mL, 60 ◦ C, 30 min), water (10× 50 mL), acetone (2× 50 mL), ether (2× 50 mL) and then dried at room temperature for 16 h.

2.4.

Preparation of sensor disk

Sieved (25 ␮m) indicator-immobilized amino-cellulose fiber (0.5 g) was stirred 16 h into a D4 hydrogel dispersion (9.5 g) containing 10% solids in 90% (w/w) ethanol/water. The resulting dispersion was knife-coated onto a 125 ␮m polyester sheet such that the indicator layer dried to a thickness of 10 ␮m. A second hydrogel layer consisting of 3% (w/w) carbon black (Degussa Corporation) in the same hydrogel dispersion was then knife-coated and allowed to dry overnight. A sensor disk 25 mm in diameter was then punched out and soaked in buffer for at least 16 h prior to use.

2.5.

Syntheses

The synthesis of the calcium fluoroionophore (8) from commercially available compounds is illustrated in Fig. 2: 2,5-Diethoxybenzaldehyde (1) was condensed with nitromethane then reduced to phenethylamine (3). Nitration and reduction of (3) yields 4-amino-2,5-diethxyphenethylamine (5), which was coupled with 4-chloronaphthalimide (9) (prepared from 4-chloronaphthalic anhydride in two steps) to form the precursor (6). Alkylation of (6) gives the t-butyl ester of the fluoroionophore in the immobilization side and ethyl ester in the binding side. De-protection of the t-butyl ester afforded carboxylic acid derivative 8 suitable for immobilization onto a solid support. The ethyl ester was removed after immobilization to provide active binding site for calcium ion. 2,5-Diethoxy-␤-nitrostyrene (2). A suspension of 40.2 g (207 mmol) 2,5-diethoxy-benzaldehyde (1), 160.14 g (2.07 mol) nitro-methane, 129.95g (2.13 mol) ammonium acetate were suspended in acetic acid (420 mL) was warmed slowly to 80 ◦ C for 20 min and kept at this temperature for 3 h. The mixture was poured into icy water (2.5 L) after cooling to room temperature. The resultant precipitate was dissolved in CHCl3 (1.2 L) and washed with water (1.2 L), saturated NaCl solution (1.2 L). The solvent was evaporated to afford 52.2 g dark reddish oil. This crude product was purified with silica gel column to afford 40.7 g (83%) yellow prism crystal (mp 55–56 ◦ C). 1 H NMR (300 MHz, CDCl3 ): ı = 1.49 (m, 6H, –CH3 ), 4.02 (m, 4H, –OCH2 ), 6.98 (m, 3H, Ar-H), 7.90 (d, 1H, CH CH-NO2), 8.10 (d, 1H, Ar-CH CH-NO2 ). Anal. Calcd. for C12 H15 NO4 : C, 60.75; H, 6.37; N, 5.90. Found: C, 60.43; H, 6.29; N, 5.98. 2,5-Diethoxyphenethylamine (3). To a suspension of 65.5 g (1.73 mol) lithium aluminum hydride in THF (2.5 L) was added 41.11 g compound 2 in THF (200 mL) during 1.5 h. The mixture was continued to heat to reflux for 4 h, cooled with

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ice-water bath to ∼15 ◦ C, and then quenched with 18% NaOH. The precipitate was filtered off and washed with THF (3× 1000 mL). The filtrate was evaporated to dryness and the residue was dissolved in CHCl3 (200 mL). This CHCl3 solution was extracted with 1N HCl (3× 200 mL). The aqueous extraction was basified with 18.3% NaOH to pH > 12, and then extracted with CHCl3 (3× 200 mL), dried over K2 CO3 . The solvent was evaporated to afford 31.11 g light yellow oil, which was distilled at 143–146 ◦ C at 1 mmHg using Kugelrohr apparatus to afford 26.1 g clear oil (72%). 1 H NMR (300 MHz, CDCl3 ): ı = 1.40 (m, 6H, –CH3 ), 2.70 (t, 2H, ArCH2 ), 2.90 (t, 2H, CH2 N), 3.98 (m, 4H, –OCH2 ), 6.70 (m, 3H, Ar-H). Anal. Calcd. for C12 H19 NO2 : C, 68.87; H, 9.15; N, 6.69. Found: C, 69.33; H, 9.24; N, 6.58. 4-Nitro-2,5-diethoxyphenethylamine (4). A solution of 50.4 g (241.3 mmol) of compound 3 in 4.0 M HCl (100 mL) was added slowly to a 35% HNO3 (620 mL) within about 2 h. The resultant yellow precipitate was stirred at room temperature for additional 20 h. The precipitate was filtered and dissolved in water (300 mL). The aqueous solution was basified to pH 12 using 40% NaOH, and extracted with CHCl3 (3× 400 mL). The organic solution was dried over K2 CO3 . The solvent was evaporated to afford 55.3 g orange oil, which solidified when cooled. This oil was purified with a silica gel column, yielding 46.8 g (78%) red oil that crystallized when cooled. 1 H NMR (300 MHz, CDCl3 ): ı = 1.45 (m, 6H, –CH3 ), 2.60 (t, 2H, ArCH2 ), 2.95 (t, 2H, CH2 NH2 ), 4.05 (q, 2H, –OCH2 ), 4.15 (q, 2H, –OCH2 ), 6.95–7.35 (m, 3H, Ar-H). Anal. Calcd. for C12 H18 N2 O4 : C, 56.68; H, 7.13; N, 11.02. Found: C, 57.23; H, 7.21; N, 10.88. 4-Amino-2,5-diethoxyphenethylamine (5). A suspension of 46.2 g (181.7 mmol) compound 4 and 2.30 g palladium on activated carbon in ethanol (300 mL) was hydrogenated at 2.2 atm for 18 h, until no more hydrogen uptake was observed. The catalyst was filtered off and washed with ethanol (3× 300 mL). Solvent was evaporated to afford 37.13 g (91%) light yellow oil. 1 H NMR (300 MHz, CDCl ): ı = 1.45 (m, 6H, –CH ), 2.60 (t, 2H, 3 3 ArCH2 ), 2.95 (t, 2H, CH2 NH2 ), 4.05 (q, 2H, –OCH2 ), 4.15 (q, 2H, –OCH2 ), 6.35–7.30 (m, 3H, Ar-H). Anal. Calcd. for C12 H18 N2 O4 : C, 64.26; H, 8.99; N, 12.49. Found: C, 63.89; H, 9.10; N, 11.98. t-Butyl 4-[4 -(4 -amino-3 ,6 -dimethoxyphenylethylamino)1 ,8 ,-naphthalimidyl-methyl] benzoate (6). 50.55 g (225 mmol) compound 5 and 31.3 g (74.3 mmol) t-butyl 4-chloro-1,8naphthalimidylmethyl benzoate [22] and 13 mL (74.6 mmol) N,N-diisopropylethylamine were suspended in 110 mL Nmethylpyrolidinone (NMP) and heated at 90 ◦ C for 18 h. The mixture was cooled and poured into water (2 L). The resultant precipitate was filtered then dissolved in CHCl3 (800 mL) and washed with water (5× 800 mL). The organic layer was dried over Na2 SO4 , filtered and evaporated to get 63.55 g crude product. The residue was triturated with hot methanol (600 mL), filtered and washed with cold methanol (600 mL). The resultant solid was re-crystallized with hot CHCl3 , afforded 31.30g (69%) bright yellow crystal. 1 H NMR (300 MHz, CDCl3 ): ı = 1.35 (m, 6H, –CH3 ), 1.50 (s, 9H, OC(CH3 )3 , 2.90 (t, 2H, ArCH2 CH2 NH), 3.50 (t, 2H, ArCH2 CH2 NH), 4.00 (q, 2H, –OCH2 ), 4.15 (q, 2H, –OCH2 ), 5.40 (s, 2H, Ar-CH2 N) 6.35–8.75 (m, 11H, Ar-H). Anal. Calcd. for C36 H39 N3 O6 : C, 70.92; H, 6.45; N, 6.89. Found: C, 70.53; H, 6.30; N, 7.01. 2-Chloroethoxyacetic acid (11). 118 mL (1.12 mol) 2chloroethoxyethanol (10) was added slowly into conc. HNO3

(70%) (625 mL) at 55 ◦ C during 8 h time period. The solution was stirred at RT for additional 18 h and heated in boiling water bath for 1 h to complete the reaction. The solution was cooled, poured into icy water (500 mL). The diluted solution was extracted with CHCl3 (5× 1 L). All extractions were combined and dried over Na2 SO4 , Solvent was evaporated to afford 83.6 g (54%) oil. This oil was used directly for next esterification without further purification. 1 H NMR (300 MHz, CDCl3 ): ı = 3.72 (t, 2H, –CH2 Cl), 3.80 (t, 2H, CH2 O), 4.25 (s, 2H, OCH2 COOH), 10.40 (s, br. 1H, COOH). Ethyl 2-chloroethoxyacetate (12). A solution of 81.6 g (590 mmol) compound 11 in 575 mL absolute ethanol containing 1 mL conc. H2 SO4 was heated under reflux for 18 h. Most of ethanol was evaporated, and the residue was dissolved in CHCl3 (600 mL) and washed with saturated NaHCO3 (3× 600 mL), dried over Na2 SO4 . The solvent was evaporated to afford 73.5 g (75%) clear oil. 1 H NMR (300 MHz, CDCl3 ): ı = 1.25 (t, 3H, –CH3 ), ı = 3.72 (t, 2H, –CH2 Cl), 3.80 (t, 2H, CH2 O), 4.15 (s, 2H, OCH2 COOH), 4.20 (q, 2H, COOCH2 CH3 ). Anal. Calcd. for C6 H11 ClO3 : C, 43.26; H, 6.65. Found: C, 43.01; H, 6.81. t-Butyl 4-{4 -[4 -(bis-ethoxycarbonylmethoxyethyl-ami  no)-3 ,6 -diethoxyphenylethyl-amino]-1 ,8 ,-naphthalimidylmethyl} benzoate (7). A suspension of 54.54 g (89.5 mmol) of compound 6, 47.5 g (285.1 mmol) of compound 11, 50.59 g (366 mmol) of K2 CO3 and 30.06 g (181 mmol) of KI in DMF (600 mL) was heated at 80 ◦ C for 20 h under nitrogen atmosphere. Then 72.6 g of compound 11 and 65.05 g of K2 CO3 were added again. The mixture was heated for 30 more hours. The solvent was evaporated and the residue was dissolved in CHCl3 (1.2 L) and water (1.2 L). The organic phase was washed with saturated NaCl and dried over Na2 SO4 . The solvent was evaporated and residue was purified with silica gel column. 22.4 g orange gum (29%) was isolated. 1 H NMR (300 MHz, CDCl3 ): ı = 1.25 (m, 6H, –COOCH2CH3 ), ı = 1.35 (m, 6H, -ArOCH2 CH3 ), 1.50 (s, 9H, OC(CH3 )3 , 2.92 (t, 2H, ArCH2 CH2 NH), 3.55 (t, 2H, ArCH2 CH2 NH), 4.00 (q, 2H, –ArOCH2 CH3 ), 4.15 (q, 2H, –ArOCH2 CH3 ), 4.20 (t, 4H, COOCH2 CH3 ) 5.40 (s, 2H, Ar-CH2 N) 6.35 -8.75 (m, 11H, Ar-H). FABMS (70 eV, mnitrobenzyl alcohol dispersion with LiI): 871 (100%) (M+H+ ); 680 (17%) (de-benzylated + H+ ). Anal. Calcd. for C48 H59 N3 O12 : C, 66.27; H, 6.84; N, 4.83. Found: C, 66.03; H, 6.81; N, 4.78. 4-{4 -[4 -(Bis-ethoxycarbonylmethoxyethylamino)-3 ,6 diethoxy-phenylethylamino]-1 ,8 ,-naphthalimidylmethyl} benzoate (8). 87.5 mL (1.14 mol) of trifluoroacetic acid (TFA) was added into a solution of 18.81 g (21.64 mmol) of compound 7 in CH2 Cl2 (160 mL). The resulting solution was stirred at for about 1 h at room temperature when the TLC indicated that most of compound 7 was gone. The mixture was then diluted with 1:1 CHCl3 :MeOH (1.2 L) and the solvent was evaporated. Repeated 6 times to remove TFA then placed on pump for 30 min to dry completely, afforded 23.2 g (103%) red gum. 1 H NMR (300 MHz, CDCl3 ): ı = 1.30 (m, 6H, –COOCH2CH3 ), ı = 1.40 (m, 6H, –ArOCH2 CH3 ), 2.92 (t, 2H, ArCH2 CH2 NH), 3.55 (t, 2H, ArCH2 CH2 NH), 4.00 (q, 2H, –ArOCH2 CH3 ), 4.15 (q, 2H, –ArOCH2 CH3 ), 4.20 (t, 4H, COOCH2 CH3 ) 5.40 (s, 2H, Ar-CH2 N) 6.35–8.75 (m, 11H, Ar-H). Anal. Calcd. for C46 H52 F3 N3 O14 as TFA salts: C, 59.54; H, 5.65; N, 4.53. Found: C, 57.53; H, 5.81; N, 4.33.

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3.

Results and discussion

3.1.

Selection of ionophore

The selection of a suitable ionophore was driven by several design criteria: (A) must contain tertiary nitrogen that can act as an electron donor and will also interact with a bound calcium cation; (B) binding properties should be insensitive to pH changes in the clinically important range of 6.6–7.8 so as to minimize undesirable pH interference to the measurement of calcium; (C) should preferentially bind calcium with a dissociation constant (Kd ) in the aqueous medium near the desired measuring range of 0.3–2.0 mM, while also in the presence of typical blood concentrations of other cations such as magnesium (0.6 mM), sodium (145 mM), potassium (4.5 mM), and lithium (<2 mM); (D) must possess an adequate chemostability during the wet storage, a minimum of 6 months at room temperature. There was a tremendous amount of calcium ionophores (ligands) reported in the literature [21]. The most famous one was bis(o-aminophenoxy)ethane-N,N,N N -tetraacetic acid (BAPTA, Fig. 1A) [8], which is the aromatic version of ethylene glycol bis-aminoethyl-tetraacetic acid (EGTA). This aromatization facilitated monitoring the calcium optically and reduced or eliminated the interference of physiological pH. The excellent selectivity against magnesium remained while the stability in the aqueous solution seemed to be worsened. It was reported that the free acid form of BAPTA was not stable for more than a few days [19], yet it was still reasonably stable for intra-cellular applications. In order to adopt BAPTA for the extra-cellular application because of its attractive selectivity, the binding strength needs to be reduced, in other words, to increase Kd value from the micro-molar to the milli-molar range. The straightforward approach was to put electron-withdrawing groups such as fluoro, chloro, bromo, or nitro at the phenyl ring to reduce the binding. The difluoro-BAPTA was chosen because of its highest fluorescence quantum yield. Unfortunately, several months of efforts were spent with little success due to some synthetic difficulties. This approach needed to be reconsidered. It did not sound rational to prepare a complicated molecule and try to simplify it again to meet a simple requirement. A simpler candidate, e.g. half of the BAPTA, should be taken into account. Following this logic, the half of the BAPTA, anisine-N,N-diacetic acid was prepared according Ref. [17] and its dissociation constant was found to be in the milli-molar range[17]. The compound C (Fig. 1) was prepared according to the similar route shown on Fig. 2, immobilized onto aminocellulose, dispersed into a hydrogel, and cast into a foil. This foil shows adequate slope (defined as percentage fluorescence signal change per milli-molar calcium, about 20%/mM) and selectivity against magnesium (30/1), but the foil was gradually loosing its slope to half of the beginning value even stored in 4 ◦ C. It may be attributed to the de-alkylation of acetic acid moiety [18,19]. That suggested that a direct linkage of acetic acid moiety to an aromatic nitrogen such as aniline should be avoided. Compound D (Fig. 1) was prepared based on this concept. The design of this compound did not seem to violate the above-mentioned design criteria (A) and (B): an aro-

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matic, instead of aliphatic tertiary amine was linked directly to a fluorophore and acts as a fluorescence quencher. The aliphatic tertiary amine was far away from the fluorophore and should not be involved in fluorescence quenching. Unexpectedly, this sensor showed strong pH dependence near the neutral pH although an adequate response to calcium in the milli-molar range. The fact that the binding of calcium causes de-protonation of the aliphatic nitrogen was believed to be responsible for such pH dependence. This ionophore had to be abandoned. So far, two things have been learned: first, no direct linkage of acetic acid to aromatic nitrogen such as aniline nitrogen; second, no aliphatic nitrogen anywhere in the molecule. There must be a way to improve the storage stability without altering the binding strength and selectivity against magnesium. The excellent storage stability of our sodium sensor [23] based on phenylazacrown ether enlightened us on a bridge of oxyethylene moiety. The addition of oxyethylene between aniline nitrogen and acetic acid moiety increases the storage stability dramatically while the binding strength and selectivity against magnesium remains almost unchanged (Figs. 1E, 3 and 4). However, the binding of compound E to calcium was found to be still too strong with the dissociation constant (Kd ) 0.2 mM [22], while the typical concentration of ionized calcium in blood is 1.2 mM. Replacing methoxy by ethoxy (Fig. 1F) did weaken the binding five times, moving Kd from 0.2 to 1.1 mM, which was close enough to the typical physiological concentration. An additional ethoxy group (Fig. 1G) was just used as a blocking group for the purpose of synthesis during the nitration of 2,5-diethoxyphenethylamine (compound 3 in Fig. 2). Fig. 3 shows the absorption spectra of compound F (Fig. 1) in water containing different concentrations of calcium chloride in the presence of 150 mM sodium chloride and 5 mM potassium chloride. The latter two salts were used to control the ionic strength and mimic the physiological environment in the real clinical samples. From these data, the dissociation constant (Kd ) was estimated at about 1 mM, close to the desired

Fig. 3 – Absorption spectra of Calcium ionophore (compound F) in water containing 145 mM NaCl and 5 mM KCl.

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Fig. 4 – Comparison of normalized absorption at 245 nm between calcium and magnesium.

1.2 mM. Although the ionic strength dependence of the Kd was not measured experimentally, it can be expected to be minimum or negligible because the total concentration span of the electrolytes in blood sample is quite narrow [23]. Fig. 4 shows the titration curves of the absorbance of compound F at 245 nm versus the logarithm of the concentration of calcium and magnesium in milli-molar. The dissociation constant (Kd ) of magnesium is larger than 10 mM. The selectivity of calcium against magnesium can be roughly estimated around 50/1, which is close to the selectivity requirement of 60/1 for blood or sera samples [23]. In our intensive real sample testing and field trials in various hospitals, we did not see any significant error caused by the interference of magnesium [33].

3.2.

Selection of fluorophore

The selection of 4-amino-1,8-naphthalimide as a fluorophore was based on the following considerations: (A) compatible with other sensors including sodium and potassium of our existing instrumental platform [23,31]; (B) excellent thermal and photochemical stability; (C) excellent fluorescence enhancement of photo-induced electron transfer (PET) [24–30]. The detailed mechanism of PET has been well found in Ref.; (D) low sensitivity to pH because of the absence of ionizable functional groups within the physiological pH range. During the selection of the fluorophore, the compatibility with other existing sensors, namely sodium and potassium sensors, became the most decisive factor for us, since the development of the fluorescent chemosensor for calcium was a continuation of the blood electrolyte project for the OPTI-series instruments. In consideration of a better manufacturability of optical instrument, it was strongly desired that all of the fluorescent sensors being developed for the OPTI-series instrument could share the same part of the fluorophore. Undoubtedly, 4-amino-1,8-naphthalimide became the first choice. This design strategy was called the “family approach” and the “family picture” has been shown in Ref. [33]. Besides the compatibility, 4-amino-1,8-naphthalimide

showed excellent thermal and photochemical stability against hydrolysis and photo-bleaching, in comparison with those conventional fluorophores such as 2 ,7 -dichlorofluorescein and 6-chloro-7-hydroxycoumarins, which had only survived for 4 and 6 weeks respectively, when stored in the pH 7.4 HEPES at 31 ◦ C [18]. The fluorescence enhancement (FE), defined as the ratio of the fluorescence intensity in the absence and saturation of an analyte, was the key factor to be considered in terms of sensor slope. The FE value of the 4-amino-1,8naphthalimide for sodium sensor [23] was reported to be 16.7, which means that the fluorescence intensity increase 16.7 fold from the baseline (absence of sodium) when the sensor is saturated with sodium ion. For calcium ion, a divalent cation, should show theoretically larger FE value than sodium ion, a monovalent cation, if a proper ionophore could be found and linked to the 4-amino-1,8-naphthalimide in the same or a similar position. This will assure that the designed sensor shows adequate sensing slope in the desired measurement range. Thanks to the powerful electron-withdrawing property of the diimide, the pKa of the amino group in the 4-amino1,8-naphthalimide was found to be around 2.5, much lower than 4.5 for the unsubstituted naphthylamine. This makes the fluorophore very insensitive to the pH near the range of physiological samples, whose pH value varies typically between 6 and 9. As a result, 4-aminonaphthalimde was selected naturally as the flourophore.

3.3. Spectral response of the sensor with calcium chloride solutions The fluorescence emission and excitation measurements on 25 mm sensor disks were carried out in a pH 7.4, 100 mM HEPES buffer containing 145 mM sodium chloride and 4.5 mM potassium chloride at room temperature. Fig. 5 shows the fluorescence emission and the excitation spectra of a sensor disk exposed to different calcium chloride solutions. The fluorescence intensity increases substantially with increasing concentrations of calcium. The binding of the cation to the

Fig. 5 – Excitation and emission spectra of a sensor disk exposed to different calcium chloride solutions: (left: Excitation spectra, fixed emission at 550 nm, right: emission spectra, fixed excitation at 470 nm).

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caused by pH change from 6.7 to 7.8 is about 2.1%, which can be translated into the concentration of calcium ion, equivalent to about 0.06 mM (2.1/34) error in the calcium concentration, which lies outside of the specification, 0.02 mM. So it is necessary to use a measured pH to correct the reported calcium concentration. Almost no fluorescence response to magnesium was observed (step j) for up to 2 mM magnesium, which is the highest concentration found in blood or serum samples. One can see from the whole curve that stable signals are obtained in minutes and the response is reversible (step f to step g). The calibration curve is shown on the insertion. The additional information about the sensor performance can be found in reference [33].

3.5.

Fig. 6 – Dynamic response of a sensor disk to various calcium chloride concentration at pH 7.40 HEPES buffer in presence of 145 mM sodium chloride and 5 mM potassium chloride. Excitation at 470 nm, emission at 540 nm. Calcium concentration, pH and magnesium concentration are (Ca/pH/Mg, in mM for Ca and Mg): (a) 0/7.40/0; (b) 0.30/7.40/0; (c) 1.20/7.40/0; (d) 2.20/7.40/0; (e) 5.0/7.40/0; (f) 10.0/7.40/0; (g) 1.20/7.40/0; (h) 1.20/6.70/0; (i) 1.20/7.80/0; (j) 1.20/7.40/2.0. Inset: fluorescence intensity as a function of calcium concentration in milli-molar.

ionophore inhibits fluorescence quenching by the anisidine donor as expected. The fluorophore does not directly interact sterically or electronically with the cation. As a result, the excitation and emission maxima are nearly invariant with the changing calcium concentrations. This is a characteristic of a photo-induced electron transfer (PET) indicator and unlike Internal Charge Transfer (ICT) probes in which the excitation maxima of a fluorophore changes upon binding of an analyte [32].

Sensor stability

The OPTI CCA instrument employs a single-point calibration process and requires extremely stable sensors. The sensor disks must maintain their factory-barcoded response parameters including the slope and intercept of the calibration curve throughout their 11-month wet-storage period at the different temperatures (4–30 ◦ C). A control solution (OPTI Check Level 3, OCL3) containing 0.80 mM calcium along with 145 mM sodium and 4.5 mM potassium were used as a standard to control the sensor performance. The aged sensors were immersed in a buffer stored under three different temperatures, namely, 4 ◦ C refrigerator, 22 and 30 ◦ C incubator. Calcium values were measured and calculated by using the aged sensor but with initially bar-coded calibration parameters (age = 0 month) at six different time periods. The more initial calibration parameters changed during the wet storage, the larger offset of the measured calcium concentration from the standard control solution would be observed. Fig. 7 shows the storage stability of the calcium sensor in terms of the offset from the true value caused by the change of sensor property during the storage. All data points are within our specification, defined as ± 0.02 mM.

3.4. Dynamic response of the sensor to calcium, pH, and magnesium changes Fig. 6 shows the dynamic response of a sensor disk to varying calcium ion concentrations in a pH 7.4 HEPES buffer containing 145 mM sodium chloride and 4.5 mM potassium chloride at room temperature (0–3000 s), to changes in pH at 1.2 mM calcium chloride (3000–4500 s) and to changes of magnesium chloride (4500–4800 s) at 1.2 mM calcium chloride. Stable signals are generally obtained in less than 1 min. The slope of the sensor is about 34%/mM in the typical clinically significant range of 0.32–2.2 mM. This allows the OPTI CCA analyzer to provide measurement precisions better than ±0.01 mM (1 S.D.), necessary for clinical decision making. As predicted, only small changes in fluorescence intensity are observed in response to changes in pH from 7.4 to 7.8 (step g and step i). However, when the sample pH falls below pH 7.4., e.g. pH 6.7 (step h), fluorescence intensity increases slightly due to partial protonation of trigger nitrogen. The total signal change

Fig. 7 – Storage stability of calcium sensor in term of offset from the true value during 11 months storage at three different temperatures, 4 ◦ C, room temperature (22 ◦ C) and 30 ◦ C.

204

a n a l y t i c a c h i m i c a a c t a 6 1 1 ( 2 0 0 8 ) 197–204

That means that the initial bar-coded sensor parameters have been maintained very well. In other words, the sensor is very stable during the wet storage.

4.

Conclusions

We have described a new optical sensor suitable for the measurement of extracellular ionized calcium with excellent storage stability in water. The addition of oxyethylene bridge between the acetic acid and the aniline nitrogen improved the storage stability dramatically in the aqueous solution. This discovery is also applicable to the BAPTA family ionophores, so that their chemo-stability during either dry or wet storage can also be improved significantly. This calcium sensor is currently used in the OPTI Medical OPTI CCA, a commercially available whole blood analyzer.

Acknowledgement The authors would like to acknowledge numerous co-workers within AVL, Roche and Osmetech.

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