WO2016056886A1

WO2016056886A1 - A system and method for measuring a fluorescence emission signal with phase correction in phase fluorometric

Info

Publication number: WO2016056886A1
Application number: PCT/MY2015/000082
Authority: WO
Inventors: Azhar Long Sulaiman Tengku; Yusri Bin Mohamad Yusof Mohamad; Hamdan Zharfan
Original assignee: Mimos Berhad
Priority date: 2014-10-09
Filing date: 2015-10-07
Publication date: 2016-04-14

Abstract

The present invention relates to the sensing instruments and methods for measuring the phase measurement of an analyte more particularly, to a system and method for measuring a fluorescence emission signal with phase correction in phase fluorometric. One of the advantages of the present invention is that the system and method removes the effect of reflected excitation light source incident on the photodetector (216) and deriving accurate reference phase without using an optical filter or requiring matching excitation and reference/auxiliary light source modulation characteristics or matching light source driver electrical circuits. Another advantage of the system and method of the present invention is that it takes into account all components of system delays from light source drivers to the photodetector (216) output as compared to the conventional correction. Moreover, the system and method of the present invention is able to remove interfering component within the same frequency.

Description

A SYSTEM AND METHOD FOR MEASURING A FLUORESCENCE EMISSION SIGNAL WITH PHASE CORRECTION IN PHASE FLUOROMETRIC FIELD OF THE INVENTION

The present invention relates to the sensing instruments and methods for measuring the phase measurement of an analyte more particularly, to a system and method for measuring a fluorescence emission signal with phase correction in phase fluorometric.

BACKGROUND OF THE INVENTION

Phase measurement is the key in sensors based on phase fluorometric, for example, in optical dissolved-oxygen (DO) sensor. One problem in making accurate phase measurement is in establishing accurate reference phase of the excitation signal at the point of detection. The phase of the fluorescence signal from the fluorophore sensing element is measured relative to the reference phase.

Another problem is a reflected excitation signal from the fluorophore sensing element. Although the sensing element construction is completed to reduce excitation signal reflection, the fluorescence signal tends to be in the same order of magnitude relative to the reflected excitation signal. Both signals are subjected to the photodetector's photo sensitivity. When the fluorescence signal and the reflected excitation signal are added together during measurement at the photodetector, this causes error in the phase measurement. TO overcome this problem, an optical filter is introduced to reject the excitation signal wavelength but this has an effect of attenuating the fluorescent signal power further. To date, none of these technologies can improve sensitivity, selectivity and response time towards measuring a fluorescence emission signal with phase correction in phase fluorometric. The present invention provides a system and method for measuring a fluorescence emission signal with phase correction in phase fluorometric. The system and method for measuring a fluorescence emission signal with phase correction in phase fluorometric in the present invention removes interfering components within the same frequency. The present invention further provides a considerable reduction of materials with even greater efficiency and economically during operation.

SUMMARY OF THE INVENTION

The present invention provides a system for measuring a fluorescence emission signal with phase correction in phase fluorometric comprising a light source for inducing a fluorophore sensing element to produce fluorescence emission; the fluorophore sensing element reacts to a modulated excitation light to produce a modulated fluorescence emission with a phase delay corresponds to a quantity of an analyte in contact with the fluorophore sensing element; a photodetector circuit having at least a photodetector to convert lights on the photodetector into an electrical signal measurement by a phase sensitive detector; the phase sensitive detector measures each phase of the predetermined frequency of the electrical signal between the photodetector circuit and a reference oscillator wherein the reference oscillator synchronously operating the light source and the phase sensitive detector; a plurality of light guides providing a direction to the lights from the fluorophore sensing element to the photodetector; a processing module for determining quantity of analyte based on the predetermined frequency of the electrical signal detected from the photodetector; a pulse width modulation (PWM) for generating pulse width modulation (PWM) signal at a predetermined frequency comprising at least an excitation PWM module for modulating the excitation light source; characterized in that an auxiliary excitation pulse width modulation (PWM) is provided for modulating an auxiliary light source; a phase adjustment module performing a phase adjustment by tuning a pulse width and a pulse location of the auxiliary excitation pulse width modulation (PWM) relative to the excitation pulse width modulation (PWM) until an amplitude of the predetermined frequency of the electrical signal from the photodetector reaches at least near to zero wherein the predetermined frequency of the reflected auxiliary light and the reflected excitation light on the photodetector to have substantially equivalent amplitude and having opposite phase to each other.

In one of the embodiment of the present invention, the system further comprising a reference signal recovery module to perform reference signal recovery for fluorophore sensing element wherein the excitation pulse width modulation (PWM) is disabled and the auxiliary excitation pulse width modulation (PWM) is configured with the pulse width and pulse location from a latest phase adjustment and the reference signal is an opposite phase to the predetermined frequency of the electrical signal from the photodetector.

In yet another embodiment of the present invention, a measurement module to perform measurement on the fluorophore sensing element with phase adjustment wherein a fluorescence signal is a predetermined frequency of the electrical signal from the photodetector.

In another embodiment of the present invention, the excitation pulse width modulation (PWM) is configured with a predetermined pulse width and pulse location relative to a reference clock.

In yet another embodiment of the present invention, the auxiliary excitation pulse width modulation (PWM) is configured with pulse width and pulse location from the latest phase adjustment.

A method of measuring a fluorescence emission signal with phase correction in phase fluorometric comprising Performing phase adjustment further comprising the steps of pulse-width modulating an excitation light at a predetermined fundamental frequency towards a phase adjustment element; pulse-width modulating an auxiliary light at said fundamental frequency towards said phase adjustment element and tuning the auxiliary light pulse width and phase parameters until the amplitude of the fundamental frequency of the reflected signal from the phase adjustment element approaches zero

Performing reference signal recovery further comprising the steps of pulse-width modulating only the auxiliary light towards said fluorophore sensing element applying same parameters from previous phase adjustment at said fundamental frequency; deriving reference signal phase from the opposite phase of the reflected signal from the fluorophore sensing element

Performing fluorescence phase measurement further comprising the steps of pulse- width modulating an excitation light at said fundamental frequency towards said fluorophore sensing element; pulse-width modulating the auxiliary light towards said fluorophore sensing element applying same parameters from previous phase adjustment at said fundamental frequency; deriving the fluorescence phase from the phase of the signal from said fluorophore sensing element minus the reference signal phase from previous reference signal recovery whereby the effect of reflection from the excitation light has been canceled out.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

Figure 1 illustrates phase and modulation of fluorescence emission relative to modulated excitation light in accordance of the present invention.

Figure 2 illustrates a system for measuring a fluorescence emission signal with phase correction in phase fluorometric for a dissolved oxygen sensor in accordance of the present invention. Figure 3 illustrates intensity curve of the reflected excitation light (310) and the fluorescence emission (311) in accordance of the present invention.

Figure 4 illustrates photo sensitivity of an avalanche photodiode (APD) detector in accordance of the present invention.

Figure 5 illustrates a flowchart for phase correction in accordance of the present invention.

Figure 6 illustrates a flowchart for measurement on the fluorophore sensing element with phase adjustment in accordance of the present invention. DETAILED DESCRIPTIONS OF THE INVENTION

The present invention will now be described in detail in connection with specific embodiments with reference to the accompanying drawings.

The present invention provides a system for optical dissolved oxygen (DO) sensor by means of phase fluorometry or more generally frequency-domain fluorometry. In this technique, a fluorophore is in contact with an analyte, for example dissolved oxygen, and is excited with light with the intensity modulated at a predetermined frequency. The resulting fluorescence emission intensity is also modulated at the same frequency. However, the emission does not exactly follow the excitation signal but exhibits time delay and amplitude variation that are determined by the intensity decay law of the fluorophore when reacting to the concentration of the analyte through collisional quenching by the analyte. The lifetime decay of the fluorescence emission is derived from a phase shift between the excitation and emission signals. The peak-to-peak amplitude of the modulated emission is reduced relative to the modulated excitation signal, which is used to measure the lifetime decay. This is depicted in Figure 1.

The lifetime decay, τ, is related to the phase, φ, and the modulation frequency, /, as shown in the equation below. tan φ = 2n/r

The lifetime decay is also related to the modulation, m, as shown in the equation below.

Once, we have the decay lifetime the concentration of the analyte, [Q] , it is computed using the Stern-Volmer equation as shown below.

T where K is the Stern-Volmer constant and τ₀ is the decay lifetime in the absence of the analyte.

The system for measuring a fluorescence emission signal with phase correction in phase fluorometric of the present invention particularly in dissolved oxygen (DO) sensor is illustrated in Figure 2. This system comprises of two pulse width modulation (PWM) modules (210,212) wherein one of the pulse width modulation (PWM) module (210) to produce excitation light source while another pulse width modulation (PWM) module (212) to produce an auxiliary light source. Light guides (214) are provided in the system of the present invention to channel lights from both light sources to a phase correction cap (223) or a sensor cap (224) the latter comprising fluorophore sensing element.

The fluorophore sensing element reacts to the modulated excitation light to emit modulated fluorescence emission of different wavelength with a phase delay to the reaction of the fluorophore on the analyte. Phase adjustment element in the phase correction cap (223) which is introduced in system of the present invention during phase adjustment condition is in place of the fluorophore sensing element that guides, mainly through reflection and proportion of light energy from each light source to the photodetector (216) with no fluorescence emission. Additional light guide is provided to channel fluorescence and reflected lights to the photodetector (216).

Subsequently, a photodetector circuit (218) is used to convert incoming light into electrical signal. In one of the embodiment, the photodetector circuit (218) may have different wavelength sensitivity to accept different gains (or attenuations) to different light wavelength. A phase sensitive detector (PSD) (220) derives phase and magnitude parameters of a frequency of the electrical signal from the photodetector circuit (218). The parameters from the PSD (220) are subsequently delivered to a processing module (222) to perform phase adjustment, reference signal recovery and analyte concentration measurement. Finally, a reference oscillator is provided to drive the two pulse width modulation (PWM) modules (210,212) and the phase sensitive detector (PSD) (220) to enable the PSD to lock to the frequency and phase of the signal from the photodetector circuit (218).

Phase sensitive detector (PSD) (220), which is also known as locked-in amplifier used in phase fluorometry in the system of the present invention. This phase sensitive detector (PSD) (220) makes use of the same reference clock or oscillator as used in modulating the excitation light source and recovery of a predetermined frequency of the converted electrical signal from the photodetector (216) by the phase sensitive detection process. However, the lights when reaching the photodetector comprise of both the fluorescence light emitted from the fluorophore sensor element and a portion of the excitation light primarily due to reflection. Although, a sensor is optically designed to reduce the effect of reflection, the fluorescence emission is too small and can be of the same order of magnitude relative to the reflected light. Figure 3 shows the intensity curve of the reflected excitation light (310) and the fluorescence emission (31 1 ). The converted electrical signal at the photodetector is the combination of the intensities of the reflected excitation light and the fluorescence light subject to the photo sensitivity of the photodetector. An example of a photodetector optical sensitivity curve is illustrated in Figure 4.

Based on the photo sensitivity curve above, although the electrical signal contribution of the reflected excitation light is attenuated more than the fluorescence emission, the former causes measureable phase error to the phase reading of the latter. The above phase error is illustrated by referring to the sum of two sinusoids with the same frequency but different amplitudes and phases as shown in the equation below:

sin ojt + R sin(o»t + p) = M sin(o>t + Θ)

In the above equation, the first term of the LHS is the true sinusoid normalized to amplitude 1 and phase 0 degree and the second term of the LHS is the interfering sinusoid having relative magnitude R and phase p. The RHS is the observed sinusoid with amplitude M and phase Θ, which are given by:

For example, with R = 0.1 and p = 90 degrees, a phase error of 5.7 degrees and an observed magnitude of 1.005 is computed. To remove the effect of the interfering excitation light incident on the photodetector, the system of the present invention provides an auxiliary light source of a different wavelength which do not cause fluorophore emission. The auxiliary light source is configured such that the fundamental frequency components of its light incident on the photodetector is of the same magnitude but has opposite phase (180 degrees difference) relative to that of the excitation light incident on the photodetector. Another alternative may also be used is by making the phase sensitive detector lock to the harmonic frequency. In one of the embodiment of the present invention, a non-fluorescence but sufficiently reflective element may be used in place of the fluorescence sensing element when tuning the auxiliary light source. Initially, the phase of the fundamental frequency of the auxiliary light source is nominally set to be opposite of the excitation light source. Due to different delays in the excitation with auxiliary optical and electrical paths, the phases of the excitation and auxiliary lights incident on the photodetector is not exactly opposite of each other. These different delays are due to different frequency responses of the light sources and mismatch in the light source driver circuits. To overcome this problem, the auxiliary light source magnitude is controlled until the phase sensitive detector registers the lowest magnitude. This technique is justified by referring again at the above equation for the sum of sinusoid with different phases and finding the minimum magnitude, i.e.:

dM²

—— = 2R + 2 cos p = 0

dR ^H

R = - cosp

In general, the minimum magnitude will not be zero and it is indicative of the phase difference between the two signals. The negative value for the magnitude has been taken care of in the above procedure by selecting opposite starting phase at the launching sites. The auxiliary light source phase is now fixed and subsequently tuned the magnitude to zero. At this point the fundamental frequency of the auxiliary light incident on the photodetector is of the same magnitude and of opposite phases. This method does not require the excitation and auxiliary light sources to have a matching frequency response characteristic.

To enable the tuning of the phase and magnitude of the fundamental frequency component (or another selected frequency) of the auxiliary light signal incident on the photodetector, the system and method of the present invention provide a solution by driving the auxiliary light source with PWM signal at the same frequency as the PW signal applied on the excitation light source. The phase (pulse location) and width of the auxiliary PWM signal is tuned relative to the excitation PWM signal until the magnitude of the fundamental frequency component (or another selected frequency) of reflected auxiliary light incident on the photodetector is the same and has opposite phase relative to the fundamental frequency component of the reflected excitation light incident on the photodetector. It is known that a periodic signal with period r(i.e., frequency ωΟ = 2τι/ Τ) can be described using Fourier series:

f{t) = a₀ + [a_n cos(n<*>₀t) + b_n sin(na>₍ 0] where

/(t) sin(nw₀t) dt ,n > 1

For a PWM signal of width T_p and period r eentered at time 0 and normalized to amplitude 1 the b„ coefficients would be zero while the a„ coefficients reduce to:

ηπ

For a preferred embodiment of the present invention, the phase sensitive detector is set to lock on the fundamental frequency of the electrical signal from the photodetector (n=1 ). The amplitude of the fundamental frequency component is given by coefficient a^

As such the amplitude of the fundamental frequency component of the PWM signal is controlled, from 0 to 1/π, by changing its duty cycle (Tp T). Note that maximum amplitude is attained at half duty cycle.

It is now described the phase adjustment procedure to correct phase error due to excitation light incident on the photodetector by first replacing the fluorescence sensor element with the phase adjustment element as illustrated in Figure 5. The phase adjustment element, mainly through reflection, delivers proportion of light energy from each of the excitation and the auxiliary light sources to the photodetector with no fluorescence emission. Then, the phase sensitive detector is configured to lock on the fundamental frequency of the PWM signals driving the light sources. A PWM signal is driven on the excitation light source with predetermined duty cycle to generate high enough optical power to allow fluorescence emission but low enough not to damage the fluorescence sensing element.

Meanwhile, another PWM signal is driven on the auxiliary light source further comprising the steps below:

a) setting the nominally phase (pulse position) of the auxiliary PWM signal 180 degrees away from the excitation PWM signal;

b) lowering the auxiliary PWM duty cycle from a predetermined initial value until the lowest amplitude reading is achieved from the phase sensitive detector. Due to imperfection in the system the reflected auxiliary light incident on the photodetector will not have exactly inverted phase relative to the reflected excitation light. The phase sensitive detector will also indicate the phase of the combination of the auxiliary and excitation light source signals incident on the photodetector;

c) Tuning the phase of the auxiliary PWM signal to attain a phase reading from the phase sensitive detector as close to zero as possible;

d) Reducing the duty cycle of the auxiliary PWM signal to attain the lowest amplitude reading possible from the phase sensitive detector;

e) Repeating the two steps above until the readings are within the required tolerance or the duty cycle reaches a system limit.

The measurement procedure incorporating the fluorophore sensing element is described below and illustrated in Figure 6:

a) Firstly, to retrieve the reference signal, the excitation light source is disabled; the auxiliary PWM signal is driven on the auxiliary light source using the duty cycle and phase from previous phase adjustment procedure. The fluorophore sensing element does not generate fluorescence emission from the auxiliary light signal. The light incident on the photodetector is therefore primarily from the reflection of the auxiliary light. b) Capturing the phase reading at the phase sensitive detector output. This is the phase of the reflected auxiliary light incident on the photodetector. The reference signal has a phase opposite that of the phase reading.

c) To perform phase measurement of the fluorescence emission signal, the excitation PWM signal is driven on the excitation light source. The excitation light causes fluorescence emission. Each part of the reflected excitation and auxiliary lights are substantially of equal amplitude and opposite phase and both cancel each other. As such the phase sensitive detector predominantly reads the phase of the fluorescence emission from the electrical signal from the photodetector.

d) Capturing the phase reading at the phase sensitive detector output. This is the phase from the fluorescence emission signal as read through the phase sensitive detector; e) Recording the relative phase between the fluorescence emission signal and the reference signal for subsequent processing, for example, averaging, conversion and displaying.

One of the advantages of the present invention is that the system and method removes the effect of reflected excitation light source incident on the photodetector and deriving accurate reference phase without using an optical filter or requiring matching excitation and reference/auxiliary light source modulation characteristics or matching light source driver electrical circuits. Another advantage of the system and method of the present invention is that it takes into account all components of system delays from light source drivers to the photodetector output as compared to the conventional correction. Moreover, the system and method of the present invention is able to remove interfering component within the same frequency.

The foregoing embodiment and advantages are merely exemplary and are not to be construed as limiting the present invention. The description of the embodiments of the present invention is intended to be illustrative and not to limit the scope of the claims and many alternatives, modifications and variations will be apparent to those skilled in the art.

Claims

1. A system for measuring a fluorescence emission signal with phase correction in phase fluorometric comprising a light source for inducing a fluorophore sensing element to produce fluorescence emission; the fluorophore sensing element reacts to a modulated excitation light to produce a modulated fluorescence emission with a phase delay corresponds to a quantity of an analyte in contact with the fluorophore sensing element; a photodetector circuit (218) having at least a photodetector (216) to convert lights on the photodetector into an electrical signal being measured by a phase sensitive detector; the phase sensitive detector measures the phase of the predetermined frequency of the electrical signal between the photodetector circuit and a reference oscillator wherein the reference oscillator synchronously operating the light source and the phase sensitive detector; a plurality of light guides (214) providing a direction to the lights to the fluorophore sensing element and the photodetector(216); a processing module (222) for determining quantity of analyte based on the predeterminedfrequency of the electrical signal detected from the photodetector(216); a pulse width modulation (PWM) (210) for generating pulse width modulation (PWM) signal at a predetermined frequency comprising at least an excitation PWM module for modulating the excitation light source; characterized in that an auxiliary excitation pulse width modulation (PWM) (212) is provided for modulating an auxiliary light source; a phase adjustment module performing a phase adjustment employing the phase adjustment element by tuning a pulse width and a pulse location of the auxiliary excitation pulse width modulation (PWM) (212) relative to the excitation pulse width modulation (PWM) until an amplitude of the predetermined frequency of the electrical signal from the photodetector reaches closest to zero wherein the modulated reflected auxiliary light and the reflected excitation light on the photodetector (216) to have substantially equivalent amplitude and having opposite phase to each other.

2. The system as claimed in Claim 1 further comprising a reference signal recovery module to perform reference signal recovery from fluorophore sensing element wherein the excitation pulse width modulation (PWM)(210) is disabled and the auxiliary excitation pulse width modulation (PWM)(210) is configured with the pulse width and pulse location from a latest phase adjustment and the recovered reference signal phase being opposite of the electrical signal phase from the phase sensitive detector (220).

3. The system as claimed in Claim 2 further comprising a measurement module employing the fluorophore sensing element wherein the excitation PWM and auxiliary PWM are both enabled, the auxiliary PWM being configured with the pulse width and pulse location from the output of the phase adjustment module, the measured fluorescence phase being the phase from the phase sensitive detector (220) minus the recovered reference signal phase.

4. A method of measuring a fluorescence emission signal with phase correction in phase fluorometric comprising

Performing phase adjustment further comprising the steps of pulse-width modulating an excitation light at a predetermined fundamental frequency towards a phase adjustment element; pulse-width modulating an auxiliary light at said fundamental frequency towards said phase adjustment element and tuning the auxiliary light pulse width and phase parameters until the amplitude of the fundamental frequency of the reflected signal from the phase adjustment element approaches zero;

Performing reference signal recovery further comprising the steps of pulse-width modulating only the auxiliary light towards said fluorophore sensing element applying same parameters from previous phase adjustment at said fundamental frequency; deriving reference signal phase from the opposite phase of the reflected signal from the fluorophore sensing element;and