Indicator Displacement Assays (IDAs): An Innovative Molecular Sensing Approach

Background and Basic Concepts of Indicator Displacement Assays

Ishfaq Ahmad Rather¹, *, Rashid Ali¹

¹ Organic and Supramolecular Functional Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, Okhla, New Delhi-110025, India

Abstract

Taking a step forward from the indicator spacer receptor (ISR) method comprising covalent linkages between receptors and indicators via a spacer, the indicator displacement assay (IDA) offers an innovative and powerful sensing approach for various target analytes in the realm of host-guest chemistry. In this chapter, we have assembled the background and conceptual details in order to give essence to the readers about this innovative sensing approach. The photophysical phenomenon and diverse non-covalent interactions involved in the sensing mechanism have been detailed. We have elucidated the need and urgency to replace the ISR approach with IDA, one having numerous advantages. The evolutionary extension of IDA for enzymatic conversion known as supramolecular tandem assays has also been described in this chapter. We believe that the present introductory chapter will give a better understanding to readers who are new to this field.

Keywords: Analyte, Competitive binding, Dynamic, Detection, Enantiomeric excess, Enzyme, Fluorescence quenching, Guest, Host, Indicator displacement assay, Indicators, Macrocycle, Non-covalent interaction, Receptors, Reversible, Recognition, Sensors, Signal, Substrate, Tandem assays.

* Corresponding author Ishfaq Ahmad Rather: Organic and Supramolecular Functional Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, Okhla, New Delhi-110025, India;

1. INTRODUCTION

Inspired by nature-based specific and selective host-guest interactions, supramolecular researchers are imitating and exploiting these interactions in molecular recognition and self-assembly phenomena. This in turn gives birth to supramolecular sensors and receptors, which play a significant role in the advancement of biological, industrial, environmental and agricultural sciences [1, 2]. In order to detect and sense a target analyte, these supramolecular sensors and receptors are taking the help of some facile sensing techniques [3, 4]. Among diverse sensing assays, the indicator displacement assay (IDA) centered on competitive experiments employing various indicators are the most acknowledged in analytical and supramolecular chemistry [5, 6]. Studies have revealed that in between receptor and indicator or analyte, both covalent as well as non-covalent

interactions exist. In fact, covalent bonds of reversible nature are extensively used in IDAs and have found widespread use in the construction of dynamic combinatorial libraries. For example, vicinal diols and aryl boronic acids interact via reversible covalent bonds, thereby leading to the formation of cyclic boronate esters. Similarly, dynamic imine centre generation between the host containing carbonyl groups and primary amine also reveals reversible covalent interactions. The various non-covalent interactions between the receptor and indicator or analyte include H-bonding, coordinate bonding, salt bridge, electrostatic interactions, anion-π interactions, cation-π interactions, hydrophobic interactions, and π-π stacking [7]. Although, the credit of introducing IDA based competitive approach in the supramolecular chemistry goes to Inouye and coworkers after carrying out the work on the recognition of acetylcholine in protic media by a supramolecular receptor [5]. Nevertheless, these competitive sensing experiments were promoted by Anslyn and teammates, after their extensive and comprehensive exploration of numerous indicators with diverse receptors for the detection of vital analytes [8, 9]. Though, IDAs have been employed to sense and detect anions as well as cations. Nonetheless, most of the IDAs have been used for anions. This is by virtue of the fact that anions are ubiquitous in nature and play central roles in several phenomena, comprising biological routes like protein biosynthesis, DNA regulation, transport of hormones, and enzyme activity. Taking these facts into consideration, sensing or recognition of anions has become a current objective of molecular recognition. Additionally, these facts have motivated chemists to bestow substantial efforts in the design and construction of real-world chemosensors for the qualitative and quantitative detection of several anions [8]. IDA experiments have shown a promising role in exploring the interactions between biological hosts (e.g. proteins, nucleic acids etc.) and various small molecules of interest, which are otherwise difficult to achieve through other sensing methodologies [10]. For example, these have been used successfully to measure helicase activity of DNA, characterize double-stranded DNA binding with RecA protein, detect triplex DNA stabilizers, selectively detect amantadine drug in saliva and human urine, and detect cocaine and sensing of caffeine, halides, and histidine [11-13]. Moreover, IDAs have also been employed to determine various analytes like inorganic phosphate, citrate, aspartate, gallic acid, nitrate, glucose-6-phosphate, heparin, tartrate, 2,3-bisphospho glycerate and inositol-1,4,5-triphosphate. Interestingly, IDAs also serve to govern the reaction kinetics of simple chemical reactions, thereby helping in understanding the mechanism of a reaction [6]. Thus, bearing in mind the real applicability of IDAs in various domains like supramolecular chemistry, bioorganic chemistry, analytical sciences, environmental and agrochemistry besides keeping in consideration the vast available literature [8, 9, 14-18], herein we have discussed the background and basic concepts of IDAs.

2. Commonly used Synthetic Receptors and Indicators for the Design of IDAs

In designing IDAs, the synthetic receptors are considered key elements. In order to fine-tune the intrinsic binding features of some familiar recognition motifs, synthetic reforms may offer highly selective and sensitive molecular constructs. Among numerous receptor systems, cyclodextrins (1), cucurbit(n)urils (2), calix [4]pyrroles (3), calix(n)arenes (5), tripodals (6), and boronic acid derivatives (7) are mostly involved in designing IDAs (Fig. 1). These supramolecular receptors are classified on the basis of capturing analytes (cations, anions, and neutral molecules), interactions (H-bonding, ion pairing/ion dipole, donor-acceptor, and π-interactions) [19-21], and structures of the hosts (macrocycles, cavitands, clefts, tweezers etc.). Owing to the structural complexity and diverse molecular conformations, these offer a promising role in the domain of molecular recognition, self-assembly, transmembrane transport, drug delivery, polymers, molecular machines, catalysis and waste water treatment [22, 23].

Fig. (1))

Some representative receptor systems (1-7) used in IDA based phenomenon.

On the other hand, these IDA-based sensors involve various colorimetric and fluorescence indicators besides others in order to signify the successful binding event with an analyte of particular interest (Fig. 2). The fluorescent and colorimetric properties of the indicators vary in free and receptor-bound form, thereby offering a change in signal upon displacement from the receptor by a desired analyte [24]. However, the choice of an indicator usually depends on the particular application being investigated and a special preference is given to naked eye color detection and Turn ON fluorescent approach. From a biological viewpoint, colorimetric indicators in certain cases are inappropriate and near infrared emitting fluorophores by virtue of higher sensitivity and deeper penetration are highly preferable. These fluorophores not only cause minimum photo damage but also lead to less background autofluorescence. Unfortunately, there is a dearth of near infrared (NIR) fluorophores for a library of synthetic receptors available, which becomes a hindrance to develop NIR-based IDAs. Here, we have provided a list of various optical indicators, which are used to design a particular IDA for the analyte of particular interest (Fig. 2).

Fig. (2))

List of some commonly used optical indicators (8-32) used in designing particular IDAs.

3. Various Physical and Chemical Phenomena Involved in Chemical Sensing

3.1. Photoinduced Electron Transfer (PET)

Photoinduced electron transfer (PET) is considered one of the most important photochemical processes for chemosensors and it takes place by means of electron exchange reactions via orbital overlap [25]. The overall PET mechanism is described as follows:

Upon photoirradiation of the donor (D) moiety, the electron gets excited from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) and develops D*. The excited electron, in turn, gets transferred to LUMO of an electron acceptor (A) moiety via frontier orbital overlap interactions and generates the radical ion-pair (D˙+-A˙‾) (Fig. 3). On the other front, the transfer of electron occurs from HOMO of A to HOMO of D* via aforesaid frontier orbital overlap interactions and thereby offering radical ion-pair (D˙‾-A˙+) (Fig. 3). As a matter of the fact that radical ion pairs involved in PET are ionic in nature and hence PET is extremely sensitive to the polarity of solvent. Thus, in order to control PET rates, the reorganization of solvent molecules surrounding ionic molecules is considered a crucial factor [26].

Fig. (3))

Schematic depiction of PET phenomenon within HOMO and LUMO molecular orbitals.

3.2. Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is regarded as a kind of energy transfer (ET) process. This ET process acts as a versatile analytical and spectroscopic technique for in vitro as well as in vivo biomolecular sensing and bioimaging besides offering a structural tool for structural interpretation of complex biomolecules [27]. Mechanistically, ET is facilitated by the dipole-dipole interaction between LUMO of excited state donor moiety (D*) and HOMO of acceptor moiety (A) (Fig. 4) [28].

Fig. (4))

Schematic illustration of energy transfer (ET) via fluorescence resonance energy transfer (FRET) mechanism.

3.3. Charge-Transfer (CT)

A charge transfer complex (CT) is formed in the ground state between the electron donor and electron acceptor moieties, thereby offering a new absorption band devoid of vibrational structure characteristics [29]. CT is mainly of two types viz. intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT). The ICT arises within a π-system possessing donor and acceptor based chromophores, while TICT is a subtype of ICT which involves a donor (D)-acceptor (A) dyads connected with each other through a single bond or vinylene linkage (Fig. 5). Upon photoexcitation, the planar D-A dyad in the ground state results in Frank-Condon state, thereby emitting from a locally excited (LE) state in a non-polar solvent. The planar LE state in a polar solvent undergoes relaxation to the twisted conformers via rotation of the connection bonds (single or vinylene bonds) in D-A pairs (Fig. 5). The twisted conformer in turn emits at longer wavelengths and leads to dual fluorescence behaviour via adiabatic photoreaction [30].

Fig. (5))

Schematic representation of the formation of TICT state via relaxation of the locally excited (LE) state.

3.4. Excited-State Intramolecular Proton Transfer (ESIPT)

The excited-state intramolecular proton transfer (ESIPT) is continuously gaining significant interest of the scientific community worldwide from past few decades [31]. The ESIPT-assisted intramolecular hydrogen bonding interaction between D and A pair results characteristic dual fluorescence of a molecule. It has been revealed that the photoirradiation of such a molecule results in a fluorescence from its enol form in the ground state (EG) through the usual Stokes shift (Fig. 6). Subsequently, the electronic distribution in the excited state enol from (EE) significantly changes and induces mutual variations in the pKa* and pKb* of the donor and acceptor units of a molecule, respectively. In the meantime, the tautomerization of EE species to the excited state keto form (EK) occurs through an extremely fast ESIPT process (Fig. 6). Thus, the formed EK emits through a large Stokes shift and decays to the ground state keto form (KG). Owing to the simultaneous recovery of pKa as well as pKb of the ground state keto form (KG), which turns back to the original ground state enol form (EG) (Fig. 6) [32].

Fig. (6))

Schematic illustration of excited-state intramolecular proton transfer (ESIPT) event.

3.5. Photon Upconversion (UC)

Photon upconversion (UC) is an interesting photophysical phenomenon in which lower energy photons are converted into higher energy photons and hence captivates much attention of the researchers working in energy related domains [33]. The UC event initiates with the excitation of the sensitizer and leads to the generation of T1 sate from S1 state via intersystem crossing (ISC) (Fig. 7). Further, through the triplet- triplet ET process, the construction of emitter T1 state from sensitizer T1 state takes place. The emitter T1 states upon encounter with each other, generates an emitter S1 state by means of triplet-triplet annihilation (TTA), and hence displays delayed fluorescence (Fig. 7) [34]. Due to deep penetration in biological cells and tissues, UC phenomenon is widely employed in the realm of in-vitro and in-vivo imaging [35].

Fig. (7))

Schematic depiction of various stages involved in the phenomenon of UC.

3.6. Aggregation-Induced Emission (AIE)

Aggregation-induced emission (AIE) is an abnormal phenomenon occurring in some organic luminophores viz. fluorescent dyes like tetraphenylethylene [36]. It is a well-established fact that the organic fluorescent dyes display emission features in the solution phase in comparison to the solid state. However, there are some organic luminophores which exhibit rotational degrees of freedom and by virtue of these rotations, such molecules upon excitation do not release energy via light to become emissive but relax back to the ground state. These luminophores undergo aggregation and become very emissive or fluorescent due to the lack of rotation. This in turn leads to an increment in photoluminescence efficiency or quantum yield [37].

4. Classification of Chemical Sensors

Chemical sensors are regarded as measurement devices which convert the chemical or physical properties of a target analyte into a detectable and measurable signal [38]. The magnitude of this measurable signal is usually proportional to the target analyte concentration. Typically, chemical sensors are made up of two components viz. sensing materials and transducers. The sensing material has a role in interaction with the target analytes (chemical stimuli), thereby resulting in a change in material property like electrical conductivity and mass. This change in material properties is then translated by the transducer into a readable (electronic) signal (Fig. 8) [39]. Chemical sensors are gifted with the potential to respond only to chemical interactions within the molecular species (chemical environment) and not to physical environments like pressure and temperature. Depending upon the signal transduction methods, chemical sensors are grouped into three major classes as:

Fig. (8))

Schematic illustration of the chemical sensor. (σ = electrical conductivity, T = temperature, ϕ = work function, m = mass, n = refractive index).

4.1. Electrical and Electrochemical Sensors

Typically, an electrical sensor is a capacitive or resistive measurement device, which is able to respond to interactions between the analyte and sensor receptor layer [40]. As a representative example, the receptors of the olfactory system of vertebrates compose an array of bioelectrical sensors. In the realm of chemical sensing, both electrical as well as electrochemical sensors are almost indistin- guishable and both comprise the interaction of various chemical analytes with an electric circuit and offer the observable or detectable response in the form of capacitance, current, resistance, and voltage. Over the past several decades, electrical and electrochemical sensors have been exploited in various fields. For instance, SnO2 based metal oxide semiconductors, conducting polymer sensors, and chemical field effect transistors (FETs) [41, 42]. Fundamentally, an electrical sensor depends on physical adsorption based primary interaction between the electrically active surface and the target chemical analytes. Unfortunately, this physical adsorption affects the sensitivity of the sensor with the change in humidity and hence creates a problem in case of laboratory or field usage. Besides, there occurs a significant drift in baseline due to