Apparatus and methods for studying bioanalytes using X-ray fluorescence

Abstract

This document discloses an apparatus comprising: an X-ray source (101); an X-ray detector (100); wherein the X-ray source (101) is configured to guide an X-ray beam (102) at a grazing angle (108) to a surface (104), at which the X-ray beam (102) is totally externally reflected by the surface (104); wherein a first bioanalyte (910) is fixed to the surface (104), and a first element (911) is attached to the first bioanalyte (910); wherein the X-ray beam (102) is capable of eliciting the emission of characteristic X-rays (109) of the first element (911); wherein the X-ray detector (100) is configured to receive the characteristic X-rays (109) of the first element (911), but not to receive the X-ray beam (102) reflected by the surface (104).

Description

[Background Technology]

[0001] X-ray fluorescence (XRF) is the emission of characteristic X-rays from materials excited by, for example, exposure to high-energy X-rays or gamma rays. If an atom is exposed to X-rays or gamma rays with photon energies greater than its electron ionization potential, electrons in the atom's inner orbitals may be ejected, leaving vacancies in those orbitals. When electrons in the atom's outer orbitals relax to fill these vacancies, X-rays (fluorescent X-rays or secondary X-rays) are emitted. The photon energy of the emitted X-ray is equal to the energy difference between the electrons in the outer and inner orbitals.

[0002] For a given atom, the possible relaxation numbers are finite. For example... Figure 1A As shown, when electrons in the L orbital relax to fill vacancies in the K orbital (L→K), the fluorescent X-ray is called K. Fluorescent X-rays from M→K relaxation are also called K. Figure 1B As shown, fluorescent X-rays from M→L relaxation are called Lα, and so on.

[0003] Analyzing fluorescence X-ray spectroscopy can identify elements in a sample because each element has characteristic energy orbitals. Fluorescence X-rays can be analyzed by classifying photon energy (energy-dispersive analysis) or by separating the wavelengths of the fluorescence X-rays (wavelength-dispersive analysis). The intensity of each characteristic energy peak is directly related to the abundance of each element in the sample.

[0004] In energy-dispersive X-ray analysis, proportional counters or various types of solid-state detectors (PIN diodes, Si(Li), Ge(Li), silicon drift detectors (SDDs)) can be used. These detectors are based on the same principle: incident X-ray photons ionize a large number of detector atoms, and the number of charge carriers produced is proportional to the energy of the incident X-ray photons. The charge carriers are collected and counted to determine the energy of the incident X-ray photons, and this process is repeated for the next incident X-ray photon. After detecting many X-ray photons, a spectrum can be constructed by counting the number of X-ray photons as a function of their energy. [Summary of the Invention]

[0005] This document discloses an apparatus comprising: an X-ray source; an X-ray detector; wherein the X-ray source is configured to direct an X-ray beam at a grazing angle to a surface, at which the X-ray beam is totally externally reflected by the surface; wherein a first bioanalyte is fixed to the surface, and a first element is attached to the first bioanalyte; wherein the X-ray beam is capable of causing the emission of characteristic X-rays of the first element; wherein the X-ray detector is configured to receive the characteristic X-rays of the first element, but not to receive the X-ray beam reflected by the surface.

[0006] According to an embodiment, the device further includes the surface.

[0007] According to an embodiment, the surface is the outer surface of the substrate.

[0008] According to an embodiment, the surface is the outer surface of a film with a thickness of less than 10 micrometers.

[0009] According to an embodiment, the membrane is a metal membrane.

[0010] According to an embodiment, the film is an epitaxial silicon film.

[0011] According to an embodiment, all X-ray beams incident on the surface from the X-ray source undergo total external reflection at the surface.

[0012] According to an embodiment, the device further includes a slit or collimator located in front of the X-ray source.

[0013] According to an embodiment, the X-ray beam guiding the surface is a fan-shaped beam.

[0014] According to an embodiment, the X-ray beam guiding the surface is a collimated beam.

[0015] According to an embodiment, the X-ray detector is configured to detect the first element by determining the photon energy of the characteristic X-ray of the first element.

[0016] According to an embodiment, the X-ray detector is configured to count the number of photons of the characteristic X-rays of the first element.

[0017] According to an embodiment, the surface is not at the interface with the liquid.

[0018] According to an embodiment, the first bioanalyte is a protein or nucleic acid.

[0019] According to an embodiment, the first element is attached to the first bioanalyte via a ligand.

[0020] According to an embodiment, the device further includes the first bioanalyte.

[0021] According to an embodiment, the device further includes a filter configured to prevent X-ray photons with energy above a threshold emitted by the X-ray source from reaching the surface.

[0022] According to an embodiment, the X-ray detector is configured not to distinguish between photons of the X-ray beam and photons of the characteristic X-rays of the first element.

[0023] According to an embodiment, the first element has a number of atoms greater than 20.

[0024] According to an embodiment, the first element has a number of atoms greater than 26.

[0025] According to an embodiment, the X-ray beam is capable of inducing the emission of characteristic X-rays of a second element of a second bioanalyte attached to the surface.

[0026] According to an embodiment, the X-ray detector is configured to receive a combination of the characteristic X-rays of the first element and the characteristic X-rays of the second element.

[0027] According to an embodiment, the X-ray detector is configured to detect the second element by determining the photon energy of the characteristic X-ray of the second element.

[0028] This document discloses a method comprising: directing an X-ray beam at a grazing angle to a surface such that the X-ray beam is totally externally reflected by the surface, wherein a first bioanalyte is fixed to the surface and a first element is attached to the first bioanalyte, and wherein the X-ray beam causes the emission of characteristic X-rays of the first element; receiving the characteristic X-rays of the first element with an X-ray detector, but not receiving the X-ray beam reflected by the surface. [Attached Image Description]

[0029] Figure 1A and Figure 1B The mechanism of X-ray fluorescence (XRF) is illustrated schematically.

[0030] Figure 2 An apparatus according to an embodiment is illustrated schematically.

[0031] Figures 3A to 3C Each schematically illustrates a cross-sectional view of an X-ray detector according to an embodiment.

[0032] Figure 4 A top view of a portion of an X-ray detector according to an embodiment is shown schematically.

[0033] Figures 5A to 5B Each schematically illustrates a component diagram of the electronic system of an X-ray detector according to an embodiment.

[0034] Figure 6 The illustration schematically depicts the time-varying current and corresponding time-varying voltage caused by charge carriers generated by incident X-ray photons according to an embodiment.

[0035] Figure 7 A flowchart of a method according to an embodiment is shown.

Detailed Implementation Methods

[0036] X-ray fluorescence (XRF) can be used to study biological samples by detecting elements attached to bioanalytes. Figure 2 A device 200 configured to study bioanalytes using an XRF with a total external reflectance (TER) configuration is schematically shown. The device 200 may include an X-ray detector 100 and an X-ray source 101.

[0037] According to an embodiment, the X-ray source 101 is configured to guide an X-ray beam 102 to a surface 104 at a grazing angle 108, at which the X-ray beam 102 is totally externally reflected by the surface 104. The grazing angle 108 is the angle between the X-ray beam 102 and the surface 104, such as... Figure 2 As shown. The upper limit of the grazing angle 108 may depend on the wavelength of the X-ray beam 102 and the characteristics of the surface 104 (e.g., material). When the grazing angle 108 is less than the upper limit, the X-ray beam 102 undergoes total external reflection. In one aspect, the X-ray source 101 is configured such that all X-ray beams from the X-ray source 101 undergo total internal reflection at the surface 104. The device 200 may include a slit or collimator 107 located in front of the X-ray source 101. The X-ray beam 102 may be a fan-shaped beam or a collimated beam. The device 200 may also include a filter 199 configured to prevent X-ray photons with energies above a threshold emitted by the X-ray source 101 from reaching the surface 104.

[0038] Surface 104 may be the outer surface of substrate 106. Surface 104 may also be the outer surface of film 105 with a thickness of less than 10 micrometers. For example, film 105 may be a metal film or an epitaxial silicon film. Surface 104 may be part of device 200. In one aspect, surface 104 is not at the interface with the liquid. In one aspect, a first bioanalyte 910 is fixed to surface 104, and a first element 911 is attached to the first bioanalyte 910. The first element 911 may be attached to the first bioanalyte 910 via a ligand. The number of atoms of the first element 911 may be greater than 20 or greater than 26. The first bioanalyte 910 may be part of device 200. The first bioanalyte 910 may be a protein or nucleic acid.

[0039] On one hand, the X-ray beam 102 enables the first element 911 to emit its characteristic X-rays 109. The X-ray detector 100 is configured to receive the characteristic X-rays 109 but not the X-ray beam 103 reflected by the surface 104, such as... Figure 2As shown. The position of the X-ray detector 100 is not necessarily fixed. For example, the X-ray detector 100 may move toward and away from the surface 104, or may rotate relative to the surface 104. In one aspect, the X-ray detector 100 is configured not to distinguish between photons of the X-ray beam 102 and photons of the characteristic X-ray 109 if the X-ray detector 100 receives photons of the characteristic X-ray beam 102. The X-ray detector 100 may be configured to detect the first element 911 by determining the energy of the photons of the characteristic X-ray 109, or may be configured to count the number of photons of the characteristic X-ray 109.

[0040] A second bioanalyte 920 fixed to surface 104 and a second element 921 attached to the second bioanalyte 920 may be present. The second bioanalyte 920 may not be spatially separated from the first bioanalyte 910. If the second bioanalyte 920 is present, the X-ray beam 102 can cause the second element 921 to emit its characteristic X-rays. The X-ray detector 100 may be arranged at approximately the same distance or different distances from the first bioanalyte 910 and the second bioanalyte 920. The X-ray detector 100 can receive a combination of characteristic X-rays from the first element 911 and characteristic X-rays from the second element 921. The X-ray detector 100 can detect the second element 921 by determining the photon energy of the characteristic X-rays from the second element 921.

[0041] Figure 3A A schematic cross-sectional view of an X-ray detector 100 according to an embodiment is shown. The X-ray detector 100 may include an X-ray absorbing layer 110 and an electronic device layer 120 (e.g., an ASIC) for processing or analyzing electrical signals generated in the X-ray absorbing layer 110 by incident X-rays. The X-ray absorbing layer 110 may include semiconductor materials such as silicon, germanium, GaAs, CdTe, CdZnTe, or combinations thereof. The semiconductor may have a high-quality attenuation coefficient for the X-ray energy of interest.

[0042] like Figure 3B A detailed cross-sectional view of the X-ray detector 100 is shown. According to an embodiment, the X-ray absorption layer 110 may include one or more diodes (e.g., pin or pn) formed by one or more discrete regions 114 of a first doped region 111 and a second doped region 113. The second doped region 113 may be separated from the first doped region 111 by an optional intrinsic region 112. The discrete regions 114 are separated from each other by either the first doped region 111 or the intrinsic region 112. The first doped region 111 and the second doped region 113 have opposite doping types (e.g., region 111 is p-type and region 113 is n-type, or region 111 is n-type and region 113 is p-type). Figure 3BIn the example, each discrete region 114 of the second doped region 113 forms a diode with the first doped region 111 and an optional intrinsic region 112. That is, in Figure 3B In the example, the X-ray absorbing layer 110 has multiple diodes, each having a first doped region 111 as a common electrode. The first doped region 111 may also have discrete portions.

[0043] When an X-ray photon strikes an X-ray absorbing layer 110 comprising a diode, the X-ray photon can be absorbed through various mechanisms and generate one or more charge carriers. The X-ray photon can generate 10 to 100,000 charge carriers. The charge carriers can drift to an electrode of one of the diodes under an electric field. This field can be an external electric field. Electrical contacts 119B can include discrete portions, each of which is in electrical contact with a discrete region 114. In embodiments, charge carriers can drift in various directions such that charge carriers generated by a single X-ray photon are substantially not shared by two different discrete regions 114 (here, "substantially not shared" means that less than 2%, less than 0.5%, less than 0.1%, or less than 0.01% of these charge carriers flow to a different discrete region 114 compared to the remaining charge carriers). Charge carriers generated by X-ray photons incident around a coverage area of ​​one of these discrete regions 114 are substantially not shared with the other of these discrete regions 114. A pixel 150 associated with discrete region 114 can be a region surrounding discrete region 114 in which substantially all (greater than 98%, greater than 99.5%, greater than 99.9%, or greater than 99.99%) of the charge carriers generated by incident X-ray photons flow to discrete region 114. That is, less than 2%, less than 1%, less than 0.1%, or less than 0.01% of these charge carriers flow through the pixel.

[0044] like Figure 3C An alternative detailed cross-sectional view of the X-ray detector 100 is shown. According to an embodiment, the X-ray absorbing layer 110 may include resistors made of semiconductor materials such as silicon, germanium, GaAs, CdTe, CdZnTe, or combinations thereof, but not diodes. The semiconductor may have a high-quality attenuation coefficient for the X-ray energy of interest.

[0045] When an X-ray photon strikes an X-ray absorbing layer 110 that includes resistors but not diodes, it can be absorbed and generate one or more charge carriers through various mechanisms. The X-ray photon can generate 10 to 100,000 charge carriers. These charge carriers can drift to electrical contacts 119A and 119B under an electric field. This field can be an external electric field. Electrical contact 119B includes discrete portions. In an embodiment, charge carriers can drift in various directions such that charge carriers generated by a single X-ray photon are substantially not shared by the two distinct discrete portions of electrical contact 119B (here, "substantially not shared" means that less than 2%, less than 0.5%, less than 0.1%, or less than 0.01% of these charge carriers flow to a different discrete portion compared to the remaining charge carriers). Charge carriers generated by X-ray photons incident on the area covered by one of these discrete portions of electrical contact 119B are substantially not shared with the other of these discrete portions of electrical contact 119B. Pixel 150 associated with a discrete portion of electrical contact 119B can be a region surrounding the discrete portion, in which substantially all (greater than 98%, greater than 99.5%, greater than 99.9%, or greater than 99.99%) of the charge carriers generated by incident X-ray photons flow to the discrete portion of electrical contact 119B. That is, less than 2%, less than 0.5%, less than 0.1%, or less than 0.01% of these charge carriers flow through a pixel associated with a discrete portion of electrical contact 119B.

[0046] Electronics layer 120 may include an electronic system 121 suitable for processing or interpreting signals generated by X-ray photons incident on X-ray absorbing layer 110. Electronics system 121 may include analog circuitry such as filter networks, amplifiers, integrators, and comparators, or digital circuitry such as microprocessors and memories. Electronics system 121 may include components shared by all pixels or components dedicated to a single pixel. For example, electronic system 121 may include an amplifier dedicated to each pixel and a microprocessor shared among all pixels. Electronics system 121 may be electrically connected to pixels via vias 131. The space between vias may be filled with a filler material 130, which may increase the mechanical stability of the connection between electronics layer 120 and X-ray absorbing layer 110. Other bonding techniques may connect electronics system 121 to pixels without using vias.

[0047] Figure 4A top view schematically illustrating a portion of an X-ray detector having an array of pixels 150 according to an embodiment is shown. This array can be a rectangular array, a cellular array, a hexagonal array, or any other suitable array. Each pixel 150 can be configured to detect X-ray photons incident thereon and determine the energy of the X-ray photons. For example, each pixel 150 is configured to detect and count the number of characteristic photons of X-rays emitted by a first element 911 and a second element 921 incident thereon within a time period, but not to count photons with energies different from the characteristic X-rays. All pixels 150 can be configured to count the number of X-ray photons incident thereon within multiple energy ranges within the same time period. According to an embodiment, detecting the characteristics of a first bioanalyte 910 based on the characteristic X-rays of the first element 911 and detecting the characteristics of a second bioanalyte 920 based on the characteristic X-rays of the second element 921 includes receiving a combination of characteristic X-rays of the first element 911 and characteristic X-rays of the second element 921, and determining the energy of the X-ray photons in this combination. Each pixel 150 may have its own analog-to-digital converter (ADC) configured to digitize an analog signal representing the energy of an incident X-ray photon into a digital signal. For XRF applications, ADCs with 10-bit or higher resolution are useful. Each pixel 150 may be configured to measure its dark current, for example, before or simultaneously with each X-ray photon incident upon it. Each pixel 150 may be configured to subtract the contribution of the dark current from the energy of the X-ray photons incident upon it. Pixels 150 may be configured to operate in parallel. For example, while one pixel 150 is measuring an incident X-ray photon, another pixel 150 may be waiting for an X-ray photon to arrive. Pixels 150 may not need to be individually addressable.

[0048] X-ray detector 100 may have at least 100, 2500, 10000 or more pixels 150. X-ray detector 100 may be configured to sum the number of X-ray photons in each interval of the same energy range counted by all pixels 150. For example, X-ray detector 100 may sum the number of pixels 150 stored in an energy range of 70 keV to 71 keV, sum the number of pixels 150 stored in an energy range of 71 keV to 72 keV, and so on. In one embodiment, characteristic X-ray photons with energies from a first element 911 and a second element 921 within a first range are counted and added to the relevant interval. X-ray detector 100 may compile the summed counts of each interval into an intensity spectrum of characteristic X-ray photons incident on X-ray detector 100.

[0049] Figure 5A and Figure 5BAll diagrams show component figures of the electronic system 121 according to an embodiment. The electronic system 121 may include a first voltage comparator 301, a second voltage comparator 302, a counter 320, a switch 305, an optional voltmeter 306, an integrator 309, and a controller 310.

[0050] According to an embodiment, a first voltage comparator 301 is configured to compare the voltage of at least one electrical contact 119B with a first threshold. The first voltage comparator 301 can be configured to directly monitor the voltage or to calculate the voltage by integrating the current flowing through the electrical contact 119B over a time period. The first voltage comparator 301 can be controllably activated or deactivated by a controller 310. The first voltage comparator 301 can be a continuous comparator; that is, the first voltage comparator 301 can be configured to be continuously activated and continuously monitor the voltage. The first voltage comparator 301 can be a clock-controlled comparator. The first threshold can be 5-10%, 10%-20%, 20-30%, 30-40%, or 40-50% of the maximum voltage that an incident X-ray photon can produce on the electrical contact 119B. The maximum voltage can depend on the energy of the incident X-ray photon, the material of the X-ray absorbing layer 110, and other factors. For example, the first threshold can be 50mV, 100mV, 150mV, or 200mV.

[0051] The second voltage comparator 302 is configured to compare a voltage with a second threshold. The second voltage comparator 302 can be configured to directly monitor the voltage or calculate the voltage by integrating the current flowing through a diode or electrical contact over a time period. The second voltage comparator 302 can be a continuous comparator. The second voltage comparator 302 can be controllably activated or deactivated by the controller 310. When the second voltage comparator 302 is deactivated, the power consumption of the second voltage comparator 302 can be less than 1%, 5%, 10%, or 20% of the power consumption when the second voltage comparator 302 is activated. The absolute value of the second threshold is greater than the absolute value of the first threshold. As used herein, the term "absolute value" or "modulus" |x| for a real number x is a non-negative value of x without regard to its sign. That is, The second threshold can be 200%-300% of the first threshold. The second threshold can be at least 50% of the maximum voltage that an incident X-ray photon can generate on the electrical contact 119B. For example, the second threshold can be 100mV, 150mV, 200mV, 250mV, or 300mV. The second voltage comparator 302 and the first voltage comparator 310 can be the same component. That is, the system 121 can have a voltage comparator that can compare the voltage with two different thresholds at different times.

[0052] The first voltage comparator 301 or the second voltage comparator 302 may include one or more operational amplifiers or any other suitable circuitry. The first voltage comparator 301 or the second voltage comparator 302 may have high speed to allow the electronic system 121 to operate under high-flux incident X-ray photons. However, having high speed typically comes at the cost of power consumption.

[0053] Counter 320 is configured to record at least the number of X-ray photons incident on pixel 150. Counter 320 may be a software component (e.g., a number stored in computer memory) or a hardware component (e.g., a 4017IC and a 7490IC). When the energy of the incident X-ray photons is determined by controller 310 to be within the interval associated with counter 320, the number recorded in the interval of counter 320 is incremented by one.

[0054] Controller 310 may be a hardware component, such as a microcontroller and a microprocessor. According to an embodiment, controller 310 is configured to begin a time delay from the time when the first voltage comparator 301 determines that the absolute value of the voltage is equal to or exceeds the absolute value of a first threshold (e.g., the absolute value of the voltage increases from below the first threshold to a value equal to or exceeding the absolute value of the first threshold). The reason for using an absolute value here is that the voltage can be negative or positive, depending on whether the voltage of the cathode or anode of a diode is used or which electrical contact is used. Controller 310 may be configured to deactivate any other circuitry not required for the operation of the second voltage comparator 302, counter 320, and the first voltage comparator 301 before the time when the first voltage comparator 301 determines that the absolute value of the voltage is equal to or exceeds the absolute value of the first threshold. The time delay may expire before or after the voltage becomes stable, i.e., the rate of change of the voltage is substantially zero. The phrase "the rate of change of the voltage is substantially zero" means that the voltage change over time is less than 0.1% / ns. The phrase "the rate of change of the voltage is substantially non-zero" means that the voltage change over time is at least 0.1% / ns.

[0055] Controller 310 can be configured to activate the second voltage comparator during a time delay period (including the start and end of the delay). In an embodiment, controller 310 is configured to activate the second voltage comparator at the start of the time delay. The term "activate" means to bring a component into an operating state (e.g., by sending a signal such as a voltage pulse or logic level, by providing power, etc.). The term "deactivate" means to bring a component into a non-operating state (e.g., by sending a signal such as a voltage pulse or logic level, by cutting off power, etc.). The operating state may have higher power consumption than the non-operating state (e.g., 10 times, 100 times, 1000 times that of the non-operating state). Controller 310 itself can be deactivated until the output of the first voltage comparator 301 activates controller 310 when the absolute value of the voltage is equal to or exceeds the absolute value of a first threshold.

[0056] The controller 310 can be configured to increment at least one quantity recorded by the counter 320 if the second voltage comparator 302 determines that the absolute value of the voltage is equal to or exceeds the absolute value of the second threshold during the time delay.

[0057] Controller 310 can be configured to cause an optional voltmeter 306 to measure the voltage upon the expiration of a time delay. Controller 310 can be configured to connect electrical contact 119B to electrical ground to reset the voltage and discharge any charge carriers accumulated on electrical contact 119B. In one embodiment, electrical contact 119B is connected to electrical ground after the time delay has elapsed. In another embodiment, electrical contact 119B is connected to electrical ground for a limited reset period. Controller 310 can connect electrical contact 119B to electrical ground via a control switch 305. This switch can be a transistor such as a field-effect transistor (FET).

[0058] In this embodiment, system 121 does not have an analog filter network (e.g., an RC network). In this embodiment, system 121 has no analog circuitry.

[0059] The voltmeter 306 can feed the voltage it measures as an analog or digital signal to the controller 310.

[0060] Electronic system 121 may include an integrator 309 electrically connected to electrical contact 119B, wherein the integrator is configured to collect charge carriers from electrical contact 119B. Integrator 309 may include a capacitor in the feedback path of the amplifier. An amplifier configured in this way is called a capacitive transimpedance amplifier (CTIA). CTIA has a high dynamic range by preventing amplifier saturation and improves the signal-to-noise ratio by limiting the bandwidth in the signal path. Charge carriers from electrical contact 119B accumulate on the capacitor during a time period (“integration period”). After the integration period expires, the capacitor voltage is sampled, and then the capacitor voltage is reset by a reset switch. Integrator 309 may include a capacitor directly connected to electrical contact 119B.

[0061] Figure 6 The diagram schematically illustrates the time-varying current (upper curve) flowing through electrical contact 119B caused by charge carriers generated by X-ray photons incident on pixel 150 surrounding electrical contact 119B, and the corresponding time-varying voltage of electrical contact 119B (lower curve). Voltage can be the integral of current relative to time. At time t0, X-ray photons strike pixel 150, charge carriers begin to be generated in pixel 150, current begins to flow through electrical contact 119B, and the absolute value of the voltage at electrical contact 119B begins to increase. At time t1, a first voltage comparator 301 determines that the absolute value of the voltage is equal to or exceeds the absolute value of a first threshold V1, controller 310 begins a time delay TD1, and controller 310 may deactivate the first voltage comparator 301 at the start of TD1. If controller 310 is deactivated before t1, controller 310 is activated at t1. During TD1, controller 310 activates a second voltage comparator 302. The term "during" as used herein refers to the start and end (i.e., the end) of a period, and any time in between. For example, controller 310 may activate the second voltage comparator 302 when TD1 expires. If, during TD1, the second voltage comparator 302 determines at time t2 that the absolute value of the voltage is equal to or exceeds the absolute value of the second threshold V2, controller 310 waits for the voltage to stabilize. The voltage stabilizes at time te when all charge carriers generated by the X-ray photons drift outside the X-ray absorption layer 110. At time ts, the time delay TD1 expires. At or after time te, controller 310 causes voltmeter 306 to digitize the voltage and determine which interval the energy of the X-ray photons falls into. Then, controller 310 causes counter 320 to increment by 1 corresponding to the number recorded in that interval. Figure 6In the example, time ts is after time te; that is, TD1 expires after all the charge carriers generated by the X-ray photon have drifted outside the X-ray absorption layer 110. If time te cannot be easily measured, TD1 can be empirically selected to allow sufficient time to collect substantially all the charge carriers generated by the X-ray photon, but not too long to avoid the risk of another incident X-ray photon. That is, TD1 can be empirically selected such that time ts is empirically determined to be after time te. Time ts does not necessarily have to be after time te, because controller 310 can ignore TD1 and wait for time te when V2 is reached. Therefore, the rate of change of the difference between voltage and the contribution of dark current to voltage is substantially zero at te. Controller 310 can be configured to activate the second voltage comparator 302 at the expiration of TD1 or at t2 or any time in between.

[0062] The voltage at time te is proportional to the amount of charge carriers generated by the X-ray photons, which is related to the energy of the X-ray photons. Controller 310 can be configured to determine the energy of the X-ray photons using voltmeter 306.

[0063] After TD1 expires or voltmeter 306 digitizes (whichever is later), controller 310 connects electrical contact 119B to electrical ground during the reset period (RST) to allow charge carriers accumulated on electrical contact 119B to flow to ground and reset the voltage. After RST, electronic system 121 is ready to detect another incident X-ray photon. If the first voltage comparator 301 has been deactivated, controller 310 can activate it at any time before the RST expires. If controller 310 has been deactivated, it can be activated before the RST expires.

[0064] Figure 7 A flowchart of a method using an XRF with a total external reflection (TER) configuration according to an embodiment is shown. In step 710, as... Figure 2 Schematic illustration: X-ray beam 102 is guided to surface 104 at a grazing angle 108, such that X-ray beam 102 is totally externally reflected by surface 104. In step 720, the characteristic X-rays 109 emitted by the first element 911 are received by X-ray detector 100, but X-ray detector 100 does not receive the X-ray beam 103 reflected by surface 104, as... Figure 2 As shown. If the second bioanalyte 920 is present on the surface 104, the X-ray detector 100 receives a combination of characteristic X-rays of the first element 911 and characteristic X-rays of the second element 921.

[0065] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, wherein the true scope and spirit are indicated by the following claims.

Claims

1. A detection device, comprising: X-ray source; X-ray detector; The X-ray source is configured to guide an X-ray beam to a surface at a grazing angle, at which the X-ray beam is totally externally reflected by the surface. The X-ray beam guiding the surface is a fan-shaped beam; In this embodiment, a first bioanalyte is fixed to the surface, and a first element is attached to the first bioanalyte. The X-ray beam can induce the emission of characteristic X-rays from the first element; The X-ray detector is configured to receive the characteristic X-rays of the first element, but not the X-ray beams reflected by the surface.

2. The apparatus according to claim 1, further comprising the surface.

3. The apparatus according to claim 1, wherein, The surface in question is the outer surface of the substrate.

4. The apparatus according to claim 1, wherein, The surface is the outer surface of a membrane with a thickness of less than 10 micrometers.

5. The apparatus according to claim 4, wherein, The membrane is a metal membrane.

6. The apparatus according to claim 4, wherein, The film is an epitaxial silicon film.

7. The apparatus according to claim 1, wherein, All X-ray beams incident on the surface from the X-ray source undergo total external reflection at the surface.

8. The apparatus of claim 1, further comprising a slit or collimator located in front of the X-ray source.

9. The apparatus according to claim 1, wherein, The X-ray detector is configured to detect the first element by determining the photon energy of the characteristic X-ray of the first element.

10. The apparatus according to claim 9, wherein, The X-ray detector is configured to count the number of photons of the characteristic X-rays of the first element.

11. The apparatus according to claim 1, wherein, The surface is not at the interface with the liquid.

12. The apparatus according to claim 1, wherein, The first bioanalyte is a protein or nucleic acid.

13. The apparatus according to claim 1, wherein, The first element is attached to the first bioanalyte via a ligand.

14. The apparatus of claim 2, further comprising the first bioanalyte.

15. The apparatus of claim 1, further comprising a filter configured to prevent X-ray photons having energy above a threshold emitted by the X-ray source from reaching the surface.

16. The apparatus according to claim 1, wherein, The X-ray detector is configured not to distinguish between photons from the X-ray beam and photons from the characteristic X-rays of the first element.

17. The apparatus according to claim 1, wherein, The first element has more than 20 atoms.

18. The apparatus according to claim 1, wherein, The first element has more than 26 atoms.

19. The apparatus according to claim 1, wherein, The X-ray beam can induce the emission of characteristic X-rays from a second element of a second bioanalyte attached to the surface.

20. The apparatus according to claim 19, wherein, The X-ray detector is configured to receive a combination of the characteristic X-rays of the first element and the characteristic X-rays of the second element.

21. The apparatus according to claim 19, wherein, The X-ray detector is configured to detect the second element by determining the photon energy of the characteristic X-rays of the second element.

22. A detection method, comprising: An X-ray beam is directed at a grazing angle to a surface such that the X-ray beam is totally externally reflected by the surface, wherein the X-ray beam directed to the surface is a fan-shaped beam, wherein a first bioanalyte is fixed to the surface and a first element is attached to the first bioanalyte, and wherein the X-ray beam causes the emission of characteristic X-rays of the first element. The characteristic X-rays of the first element are received by an X-ray detector, but the X-ray beams reflected by the surface are not received.

23. The method according to claim 22, wherein, The X-ray detector is configured to detect the first element by determining the photon energy of the characteristic X-ray of the first element.

24. The method according to claim 22, wherein, The X-ray detector is configured to count the number of photons of the characteristic X-rays of the first element.

25. The method according to claim 22, wherein, The X-ray detector is configured not to distinguish between photons from the X-ray beam and photons from the characteristic X-rays of the first element.

26. The method according to claim 22, wherein, The first bioanalyte is a protein or nucleic acid.

27. The method according to claim 22, wherein, The first element is attached to the first bioanalyte via a ligand.

28. The method according to claim 22, wherein, The first element has more than 20 atoms.

29. The method according to claim 22, wherein, The first element has more than 26 atoms.

30. The method according to claim 22, wherein, The surface is not at the interface with the liquid.

31. The method according to claim 22, wherein, All X-ray beams incident on the surface from the X-ray source undergo total external reflection at the surface.

32. The method according to claim 22, wherein, The surface in question is the outer surface of the substrate.

33. The method according to claim 22, wherein, The surface is the outer surface of a membrane with a thickness of less than 10 micrometers.

34. The method according to claim 33, wherein, The membrane is a metal membrane.

35. The method according to claim 33, wherein, The film is an epitaxial silicon film.

36. The method according to claim 22, wherein, The X-ray beam causes the emission of characteristic X-rays of a second element of a second bioanalyte attached to the surface.

37. The method of claim 36, wherein, The X-ray detector receives a combination of the characteristic X-rays of the first element and the characteristic X-rays of the second element.

38. The method according to claim 36, wherein, The X-ray detector is configured to detect the second element by determining the photon energy of the characteristic X-rays of the second element.

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