Chemistry: analytical and immunological testing – Metal or metal containing – Li – na – k – rb – cs – fr – be – mg – ca – sr – ba – ra
Reexamination Certificate
1998-09-30
2001-01-09
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Metal or metal containing
Li, na, k, rb, cs, fr, be, mg, ca, sr, ba, ra
C422S051000, C422S082050, C422S082060, C422S082070, C422S082080, C422S082090, C436S073000, C436S172000, C562S405000, C562S433000, C562S443000
Reexamination Certificate
active
06171866
ABSTRACT:
The present invention relates to luminescence indicators for determining ionized calcium in a sample as well as optical sensors comprising these luminescence indicators. The invention also relates to a method of determining calcium ions in a sample using the luminescence indicators of the invention.
BACKGROUND OF THE INVENTION
For determining the calcium ions, the sample is contacted at least indirectly with a luminescence indicator (=luminophore-ionophore) having a luminophoric moiety and an ionophoric moiety, which ionophoric moiety reacts with the calcium ions present in the sample, whereupon the luminescence of the luminophoric moiety is measured and the concentration or the activity of the ionized calcium is deduced utilizing the test reading, i.e. the calcium ion is determined.
A determination method of this type is based on the reversible binding of calcium ions to a Ca
2+
-selective ionophore and on the so-called “PET effect” between the ionophore and a luminophoric moiety.
The reversible binding of calcium ions to the ionophore proceeds according to the principle of mass action (Equation 1):
ICa
2
+
⟷
Kd
I
+
Ca
2
+
(
1
)
wherein, at a given ionic strength and temperature, the dissociation constant (K
d
) is given by Equation 2,
K
d
=
c
I
c
Ca
2
+
c
I
_
Ca
2
+
(
2
)
wherein I means the ionophore with the charge number −2, ICa the ionophore-ion complex and c the concentration. In the following, K
d
and cCa
2+
are given in mol/l and mmol/l, respectively.
The PK
d
value (Equation 3) is the negative common logarithm of the dissociation constant:
PK
d
=−log(K
d
) (3)
The term “PET effect” denotes the transfer, induced by photons, of electrons (photoinduced electron transfer=PET) from the ionophoric moiety or ionophore to the luminophoric moiety or luminophore, which causes a decrease in the (relative) luminescence intensity and the luminescence decay time of the luminophore. Absorption and emission wavelengths of the luminophoric moiety or luminophore, respectively, remain basically unaffected in the process (J. R. Lakowicz in “Topics in Fluorescence Spectroscopy”, Volume 4: Probe Design and Chemical Sensing; Plenum Press, New York & London (1994)).
By the binding of ions to the ionophore, the PET effect is reduced or completely suppressed, so that there is an increase in the luminescence of the luminophoric moiety. Hence, the concentration or the activity of the ion to be determined can be deduced by measuring the change in luminescence properties, i.e. luminescence intensity and/or luminescence decay time.
In mammals, calcium plays important physiological roles. These roles include: (1) controlling blood coagulation by activating the formation of thrombin from prothrombin, (2) excitation of muscle, heart and nerve cells, including acting as a second messenger like cAMP.
Measurement of extracellular ionized calcium is an essential part of medical diagnostics. The measurement of ionized calcium, blood gases and potassium is mandatory to allow for the maintenance of good cardiac function during liver transplant operations or other operations that require bypassing the heart and lung functions and using artificial life support.
Ion selective electrodes (ISE) have been used for determining calcium ions in body fluids for many years. Serious drawbacks of electrochemical measuring arrangements are the requirement of a reference element, sensitivity towards electrical potentials and electromagnetic interference. While ion-selective electrodes are rugged and reliable, they are expensive to use in a disposable device application. In addition, these electrodes require an electrical connection of the sample measurement device to the instrument.
However, optical methods or optical sensors do not require a reference element. The optical signals are independent of external potentials and currents. Such optical methods of determining calcium as are known to date are based f.i on the measurement of the luminescence intensity or luminescence decay time of a calcium-specific luminescence indicator or the light absorption of a calcium-specific absorption indicator, which depend directly or indirectly on the concentration or activity of calcium ions.
In order to determine intracellular calcium concentrations, indicators which change their absorption and/or luminescence properties by reversible binding of calcium ions (see above, Equation 1) are, e.g., used. Suitable indicators for intracellular calcium determination are based e.g. on tetracarboxylate Ca
2+
chelating compounds having the octacoordinate ligating group characteristics of EGTA (=ethylene glycol bis(-beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) and BAPTA (=1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid).
U.S. Pat. No. 4,603,209 e.g. discloses the BAPTA analogs which are electronically coupled to one or two fluorescent dye molecules capable of being excited in the UV range. By the binding of calcium ions to the ionophore, the absorption wavelength of the dye (a stilbene derivative) is changed. By means of suitable electron-withdrawing or electron-donating substituents it is feasible to change the calcium dissociation constant in the range of 80 to 250 nmol/l.
U.S. Pat. No. 5,049,673 further describes BAPTA analogs which—in electronically decoupled condition—are bound to xanthene dyes (fluoresceines, rhodamines). As a result of the electronic decoupling from the fluorophore, a PET effect occurs which is recognizable from the constant spectral position of the fluorescence emission band and the increasing fluorescence intensity depending on increasing concentrations of Ca
2+
(see FIG.
4
a
of the reference). From the low K
d
values it can be seen that the fluoroionophores described (see
FIG. 6
of the reference) are not useful for the determination of millimolar concentrations of Ca
2+
in whole blood or blood plasma.
As a consequence of the very low dissociation constant, such calcium-ionophores are useful for determining Ca
2+
in samples having correspondingly low calcium values, such as e.g. intracellular Ca
2+
. In contrast to this, blood plasma for example has Ca
2+
concentrations in the range of about 0.4-2 mmol/l. Suitable ionophores for optical determination of Ca
2+
in the blood or blood plasma thus must have correspondingly high K
d
values. Ideal K
d
values lie within the expected range of the Ca
2+
concentrations or activities to be determined.
From U.S. Pat. No. 5,516,911, fluorescent indicators based on fluorinated BAPTA derivatives are known which have K
d
values in the millimolar range. One major disadvantage with this method is the very complicated synthesis of fluorinated BAPTA derivatives.
Moreover, the known ionophores based on BAPTA or on derivatives thereof in an aqueous environment and at normal ambient temperatures are not particularly stable chemically (see U.S. Pat. No. 4,603,209, column 26, lines 40-46). This is particularly disadvantageous in determination procedures using optical sensors in measuring situations requiring a high shelf life (durability) of the sensor or where, for monitoring purposes, one sensor is to be used for measuring over prolonged time periods.
The present invention aims at avoiding these disadvantages and problems and has as its object to provide luminophore-ionophores for the optical determination of calcium ions, whose ionophores if compared to such BAPTA compounds as are known to date, in particular fluorinated derivatives, are more easily synthesizable and—in electronically decoupled condition—can be covalently bound to suitable luminophores.
Further, the ionophores of the provided luminophore-ionophores are to exhibit K
d
values allowing the determination of physiological calcium values without requiring previous diluting of the sample material, wherein, by means of suitable substituents, the K
d
valu
He Huarui
Mortellaro Mark Alan
AVL Medical Instruments
Baker & Botts L.L.P.
Soderquist Arlen
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