Construction of biomolecular sensors based on quantum dots

Abstract

At this post-genomic era, the focus of life science research has shifted from life genetic information to general biofunctions. Thus, the diverse biomacromolecules and biomicromolecules that are embodiments of gene functions become the hotspot of life science research. The major task is to acquire some reliable and efficient routine analytical techniques that can identify and detect these biomolecules. In particular, biomolecular sensors based on quantum dots (QDs) are a major target that can identify and detect biomolecules. This study has the following innovations: previous reviews about QD sensors have focused on their advantages in detection, and the detection targets include not only biomolecules but also non-biomolecules. In comparison, this study is focused on QD sensors targeting biomolecules, and covers not only the QD biosensors that detect biomolecules, but also other types of QD sensors that detect biomolecules. Thus, different from other reviews that are focused on QD sensors targeting biomolecules, we summarize the cons, pros and developing trends of biomolecular sensors from the perspectives of fluorescent QD biomolecular sensors, room-temperature phosphorescence (RTP) sensors, QD nanohybrids, and QD-based electrochemical biosensors. We expect to provide new clues for the development of better biomolecular sensors.

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1. Introduction of biomolecular sensors

With the intensified research on genomics and the implementation of the human genome project, the focus of life science research has shifted from life genetic information to the overall biological functions, indicating that genomics has entered the post-genomic era. In this era, a variety of large and small biomolecules, as the embodiments of gene functions, become the hotspot of life science research. Research on these biomolecules is primarily aimed at acquiring routine, reliable and efficient analytical techniques for the recognition and detection of these molecules. In analysis of real biological samples, however, various biomolecules always coexist and severely mutually interfere with matrix components, such as the background fluorescence, scattering light and metal ions in biological fluids. Thus, one bottleneck in modern life science research is how to easily and efficiently separate and recognize target biomolecules from complex organism matrix samples. Thus, it is of high practical significance to investigate and build biomolecular sensors with higher selectivity, stronger anti-interference ability and higher detection efficiency, and thus to use them to accurately and efficiently identify target biomolecules from real biological samples.

The biomolecular sensors in this study are devices used to qualitatively and quantitatively detect biomolecules. With the rapid development of nanotechnology since 1990s, research on biosensors witnessed new opportunity and enjoyed breakthrough during its integration with nanotechnology. Because of unique physiochemical properties (e.g. quantum effect, macroscopic quantum tunnelling effect, surface effect, and small size effect), nanomaterials can easily integrate with biosensors to form novel nano-biosensors with properties of nanomaterials. As a result, biosensors are endowed with higher sensitivity, higher reliability, quicker response and smaller volume, which have greatly promoted their development. Under the promotion of nanotechnology, novel nanomaterials are widely applied to construct high-sensitivity and high-precision biosensors.^1–5 So far, the high-performance sensors for identification and detection of biomolecules become a hotspot in the fields of life science and analytical chemistry.

Among diverse biomolecule recognition/detection sensors, the most important type is biosensors, which are analytical devices based on enzymes, liposome and antigen/antibody as the recognition components. Research on biosensors started with the emergence of enzymic biosensors in 1960s and developed rapidly in 1980s. So far, biosensors become the cutting-edge field in modern analytical chemistry and are widely applied in biomolecule detection, clinical medical diagnosis, environmental monitoring and other areas.^1–5 Biosensors can be divided by the sensing material into DNA/RNA biosensor, enzyme biosensor, microbial sensor, immunosensor, tissue biosensor, molecular imprinted biosensor, and organelle biosensor.^6,7 Depending on the principle of signal conversion, biosensors can be divided into optical biosensor, electrochemical biosensor, and calorimetric biosensor.

Previous reviews about QD-based sensors are focused on the exploitation of biosensors, exploitation of sensors based on the principle of energy resonance or electron-transfer, application of biological marking, and the advantages of QD-based sensors.^8–18 These reviews are focused on the advantages of QD-based sensors, and their applications in biology, medicine and environment. In other words, the focus is the characteristics of QD-based sensors, but rarely about the identification of some specific target by QD-based sensors. Moreover, there is also rare review about QD-based sensors that target at biomolecules. Thus, this study is focused on biomolecules, and particularly summarizes the characteristics of QD-based sensors in identifying and marking biomolecules (excluding non-biomolecules). We expect to provide new clues for exploitation of better biomolecular sensors.

2. Properties of quantum dots (QDs)

When the size of semiconductor materials decreases to the critical level, the three-dimensional motion of electrons will be restricted, showing the quantum confined effect, and these materials are called quantum dots (QDs). The compositional elements of QDs include II–VI family (e.g. CdSe, ZnS), III–V family (e.g. GaAs, InP) and IV–VI family (e.g. PbS, PbSe), and the most-studied elements are CdX (X = S, Se, Te).¹⁹ The most notable property of typical QDs is the size-dependent photoluminescence. For instance, CdSe@ZnS QDs show size-specific colors: when the size increases from 2.7 to 4.8 nm, the emission peak occur at 510 to 610 nm accordingly (Fig. 1A).²⁰ Therefore, the emission spectrum of QDs can be expanded from the ultraviolet zone to the near infrared zone by adjustment of type and size of QDs (Fig. 1B).^21,22 The size-specific changeability of emission spectrum is attributed to the quantum confined effect of QDs.²²

Fig. 1 Size-dependent PL characteristics of QDs. (A) Cartoon, photograph, and PL spectra of typical CdSe@ZnS QDs with increasing sizes. Reprinted with permission from ref. 9. Copyright 2011 American Chemical Society. (B) Representative QD core materials scaled as a function of their emission wavelength superimposed over the spectrum. Reproduced by permission from ref. 21. Copyright 2005, rights managed by Nature Publishing Group.

QDs outperform traditional dyes in many aspects (Table 1).^23,24

Table 1 Comparison of properties of organic dyes and QDs²⁴

Property	Organic dye	QDs
Absorption spectra	Discrete bands, FWHM 35 nm to 80–100 nm	Steady increase toward UV wavelengths starting from absorption onset; enables free selection of excitation wavelength
Molar absorption coefficient	2.5 × 10^4 to 2.5 × 10^5 M^−1 cm^−1 (at long-wavelength absorption maximum)	10^5 to 10^6 M^−1 cm^−1 at first excitonic absorption peak, increasing toward UV wavelengths; larger (longer wavelength) QDs generally have higher absorption
Emission spectra	Asymmetric, often tailing to long-wavelength side; FWHM, 35 nm to 70–100 nm	Symmetric, Gaussian profile; FWHM, 30–90 nm
Stokes shift	Normally <50 nm, up to >150 nm	Typically <50 nm for visible wavelength-emitting QDs
Quantum yield	0.5–1.0 (visible), 0.05–0.25 (NIR)	0.1–0.8 (visible), 0.2–0.7 (NIR)
Fluorescence lifetimes	1–10 ns, mono-exponential decay	10–100 ns, typically multi-exponential decay
Two-photon action cross section	1 × 10^−52 to 5 × 10^−48 cm^4 s per photon (typically about 1 × 10^−49 cm^4 s per photon)	2 × 10^−47 to 4.7 × 10^−46 cm^4 s per photon
Solubility or dispersibility	Control by substitution pattern	Control via surface chemistry (ligands)
Binding to biomolecules	Via functional groups following established protocols. Often several dyes bind to a single biomolecule. Labeling-induced effects on spectroscopic properties of reporter studied for many common dyes	Via ligand chemistry; few protocols available. Several biomolecules bind to a single QD. Very little information available on labeling-induced effects
Size	∼0.5 nm; molecule	6–60 nm (hydrodynamic diameter); colloid
Thermal stability	Dependent on dye class; can be critical for NIR-wavelength dyes	High; depends on shell or ligands
Photochemical stability	Sufficient for many applications (visible wavelength), but can be insufficient for high-light flux applications; often problematic for NIR-wavelength dyes	High (visible and NIR wavelengths); orders of magnitude higher than that of organic dyes; can reveal photobrightening
Toxicity	From very low to high; dependent on dye	Little known yet (heavy metal leakage must be prevented, potential nanotoxicity)
Reproducibility of labels (optical, chemical properties)	Good, owing to defined molecular structure and established methods of characterization; available from commercial sources	Limited by complex structure and surface chemistry; limited data available; few commercial systems available
Applicability to single molecule analysis	Moderate; limited by photobleaching	Good; limited by blinking
FRET	Well-described FRET pairs; mostly single-donor–single-acceptor configurations; enables optimization of reporter properties	Few examples; single-donor–multiple-acceptor configurations possible; limitation of FRET efficiency due to nanometer size of QD coating
Spectral multiplexing	Possible, 3 colors (MegaStokes dyes), 4 colors (energy-transfer cassettes)	Ideal for multi-color experiments; up to 5 colors demonstrated
Lifetime multiplexing	Possible	Lifetime discrimination between QDs not yet shown; possible between QDs and organic dyes
Signal amplification	Established techniques	Unsuitable for many enzyme-based techniques, other techniques remain to be adapted and/or established

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(1) Broad range of excitation wavelength and narrow range of emission wavelength. Because of this property, several types of QDs can be excited at the same exciting wavelength and emit fluorescence at different wavelengths, which enables the synchronous mark of QDs at different particle sizes.^25,26 In comparison, traditional fluorescent dyes exhibit very narrow excitation wavelength range and thus should be excited at several excitation wavelengths, which is inconvenient for practice.

(2) Large Stokes shift. The narrowness and symmetry of emission peaks and the small overlap help to avoid the overlap between the emission spectrum and the excitation spectrum, which is favorable for detection of fluorescent signals. In comparison, the emission peaks of fluorescent dyes are encountered with narrow-range, asymmetry and severe tailing, and the use of different organic dyes will lead to overlap and mutual interference of emission spectra, which complicate the practical analysis and detection.

(3) Synthesis of QDs at any wavelength. The emission wavelength can be adjusted by controlling the size and composition of QDs, and the spectral peaks of size-even QDs are in symmetric Gaussian distribution. In comparison, the emission wavelength of traditional organic fluorescent dyes is not adjustable and the peak shape is in logarithmic normal distribution.

(4) High photostability. QDs outperform fluorescent dyes with 10–20 times higher fluorescent intensity and stability; very small photobleaching,²⁷ stable photochemical properties and difficulty in bio-degradation or metabolism. With several weeks or longer duration of fluorescence, QDs can be used into dynamic observation of cells or proteins in vivo and into long-time observation of a marked object. QDs are able to endure repeated excitation without fluorescent quenching.

(5) High biocompatibility. Specific surface coating to QDs can connect required groups and be used into marking or molecular detection in vivo. In comparison, fluorescent dyes possess high toxicity and low biocompatibility.

In general, QDs are characterized by high yield of fluorescent quanta, photobleaching resistance, relatively narrow symmetrical emission spectrum, and broad continuous excitation spectrum. These properties allow QDs to be ideal material for fluorescent biosensors and a hotspot in biochemical fluorescence analysis (Fig. 2).^28,29 Especially doped quantum dots.^30–37

Fig. 2 Properties of quantum dots and their analytical applications. Reprinted with permission from ref. 29. Copyright 2011 Elsevier Ltd.

3. Fluorescent QDs biomolecular sensors

The four effects (quantum size effect, small size effect, surface effect, macroscopic quantum tunneling effect) endow nanoparticles with special physiochemical properties, which allow nanoparticles to be widely applied in medicine, catalysis and materials. An emerging application area of nanotechnology is biomolecular sensors, especially the rapidly-developing fluorescence nano-biomolecular sensors. Fluorescent nano-biomolecular sensors are able to detect several types of physiological analytes in vivo, which is unachievable by organic dyes.²⁸ Because of high sensitivity, rapid response, strong anti-interference and no requirement of reference, fluorescent nano-biomolecular sensors are widely applied in biological detection, immunoassay, biological medicine, fluorescence labeling, and in vivo imaging in recent years. Fluorescent nano-biomolecular sensors are a powerful biological analysis technology in medical diagnosis and clinical treatment. Regarding the design and application of nano-biomolecular sensors into biomedicine, the key is specific biocompatibility modification at surfaces of QDs³⁸ (Fig. 3), which will improve the water-solubility and biocompatibility of QDs.

Fig. 3 Different approaches to conjugation of quantum dots to biomolecules. Reprinted with permission from ref. 38. Copyright 2006 WILEY-VCH Verbg GmbH&Co. KGaA, Weinheim.

3.1. QDs-Based fluorescence resonance energy transfer (FRET)

FRET is a nonradiant energy transition, in which the interaction of intermolecular electric dipoles transits the excited-state energy in the donor to the excited-state receptor. For organic fluorescent dyes, the narrow absorption spectrum and the tailing of emission spectrum will cause overlap between the donor's emission spectrum and the receptor's absorption spectrum. These defects are overcome by QDs. The narrow emission spectrum and the absence of tailing in QDs avoid the overlap of donor's and receptor's emission spectra and thus prevent mutual interference. Meanwhile, because of broad excitation wavelength, QDs at different sizes can be excitated by the same wavelength, and the donor's emission wavelength and the receptor's absorption wavelength can be well overlapped, thereby ensuring the efficiency of resonance energy transfer.³⁹

Research on QDs-based FRET energy donors was started in 2003.⁴⁰ In this biomolecular sensors based on maltose-binding protein (MBP), the C-terminal of MBP binds to 5-His to form MBP-5His, which improves the coordination ability without reducing bioactivity. In this way, each QD binds to a certain amount of MBP, which leads to the formation of a QDs–MBP sensor with a constant quantum yield. When a β-cyclodextrin (β-CD)-based β-CD–QSY9 binds to the binding sites of MBP, the occurrence of FRET between QDs and QSY9 leads to the quenching of fluorescence. After β-CD–QSY9 is replaced by maltose, the fluorescence of QDs is recovered. The fluorescence intensity of this sensor is enhanced with the increase of maltose concentration, which is the basis for maltose detection. However, compared with metal chelates and organic fluorescent dyes, the QDs are relatively larger-size, which lengthens the space between donor and receptor and also restricts the efficiency of resonance energy transfer. To eliminate these impacts, QDs were coordinated with Cy3-marked MBP to form QDs–MBP–Cy3, which was then bound to the sugar receptor site in MBP to form a two-step FRET. Such treatment overcomes the restriction of space between QDs and receptor^39,40 (Fig. 4). They conducted a series of systemic further experiments and fully elaborated the advantages of QDs for application as energy donor in FRET.^41–46

Fig. 4 (A) Scheme of a 560QDs–MBP–β-CD–QSY-9 maltose biomolecular sensors assembly; (B) scheme of a 530QD–MBP–Cy3–β-CD–Cy3.5 maltose biomolecular sensors assembly. Reprinted with permission from ref. 40. Copyright 2003 Nature Publishing Group.

In terms of protein detection, red (611 nm) and green (555 nm) emission spectra were used to mark bovine serum albumin (BSA) and BSA antibody (IgG) respectively.⁴⁷ When the antigen and antibody bind, the green fluorescence of IgG-connected QDs was weakened, while the red fluorescence of BSA QDs was enhanced accordingly. The reason is that during the binding, the QDs carried by the antigen and antibody approached each other, and as a result, the excited-state energy from the smaller-size green QDs is transited to the excited state of the larger-size red QDs, or namely the occurrence of resonance energy transfer. With addition of pure BSA, the BSA competed with and replaced QD–BSA, which prevented the occurrence of resonance energy transfer. As a result, the intensity of red fluorescence was weakened, while the green fluorescence was recovered. On this basis, a BSA detection sensor with strong specificity was built.

As reported, the FRET system involving QDs and Cy5 was used to build a cocaine adapter sensor.⁴⁸ As showed in Fig. 5, with the absence of cocaine, FRET occurs between QDs and Cy5, and Cy5 blue fluorescence is detected. With the addition of cocaine, it binds to the adapter, so the FRET between QDs and Cy5 does not occur. This sensor is capable of cocaine detection depending on the changes of Cy5 blue fluorescent.

Fig. 5 Principle of signal-off single QD-based aptameric sensor for cocaine detection. Reprinted with permission from ref. 48. Copyright 2009 American Chemical Society.

QDs FRET systems with many advantages are widely applied in analysis of conformational changes of biomacromolecules,⁴³ immunoassay,^46,47 nucleic acid detection,^49–56 enzyme activity measurement,^57–60 and interaction between biomacromolecules.⁶¹

3.2. QDs-Based photoinduced electron transfer (PIET)

A PIET fluorescent sensor is composed of three parts: a fluorophore, a connector and a quencher. The fluorophore after excitation emits fluorescence, while the quencher binds to the connector on the fluorophore, and the electrons receiving the fluorophore will quench the fluorescence. With the presence of a detected substance, the quencher and the substance bind and are deprived of electron accepting ability, which leads to the recovery of fluorescence. The stimulated QDs will undergo electron or energy transfer with their donor or receptor, leading to the quenching of photoluminescence.^62–64 Such characteristic can be used into molecular recognition and into transforming the concentration signal of the analyte to detectable signal.^63,65 However, the sensors based on the principle of QDs–PIET emerged later and their detection mechanism is unclear.⁶⁵ At present, only a few PIET-based sensors have been applied in detection of glucose, maltose, DNA and anion.^66–69 Raymo et al.^63,70 proposed a PIET working mechanism based on the signal receptor-substrate: the quencher is adsorbed electrostatically onto the surface of QDs to quench the QDs; then a receptor that can combine with the quencher is added to desorb the quencher from the surface and to recover the QDs (Fig. 6). On this basis, various QDs PIET-based sensors can be built. Since the donor–receptor distance required in electron-transfer is much shorter than that in FRET,⁷¹ QDs-based electron-transfer sensors are more applicable for small-molecule detection.

Fig. 6 (A) The supramolecular association of quencher and receptor prevents the electron transfer process and activates the luminescence of the quantum dot; (B) the supramolecular association of protein and ligand prevents the electron transfer process and activates the luminescence of the quantum dot. Reprinted with permission from ref. 70. Copyright 2008 Springer.

3.3. Application of fluorescent QDs in biomedicine

The fluorescent QDs analytical method shows high luminescent performance and biocompatibility, and thus become an indispensable technique in modern drug detection. For instance, in a QDs fluorescent probe for acetylcholine detection, acetylcholine can quench the fluorescence of TOPO-modified CdSe/ZnS QDs.⁷² Antibody-coated magnetic nanoparticles (MNPs) and a QDs antibody are combined to form a fluorescent sensor for detection of Salmonella.⁷³ Choline oxidase, QDs and acetylcholine were assembled to form a biosensor for detection of organophosphorus (OP) pesticides, and this high-performance biosensor is favorable for the development of rapid and high-flux detectors of OP pesticides.⁷⁴ A novel high-selectivity fluorescent nano-biosensor was developed for detection of CN⁻ in water solutions.⁷⁵ Moreover, fluorescent QDs have also been applied into detection of biomacromolecules such as DNA,^76–78 proteins^79–81 and disease biomarkers^82,83 (Fig. 7), as well as drugs such as ciprofloxacin,⁸⁴ doxorubicin⁸⁵ and ceftriaxone sodium.⁸⁶

Fig. 7 Schematic representation of the sandwich RMFIA for the detection of disease biomarkers. Reprinted with permission from ref. 82. Copyright 2016 American Chemical Society.

3.4. Application of fluorescent QDs in biological imaging

In 1998, it was found that QDs after surface modification could be used as fluorescent bioprobes for living cells, which is the basis for development of QDs fluorescent probes. Later, red fluorescence and green fluorescence nanoparticles were used to mark fibroblasts of ³T₃ mice; the green nanoparticles bound with urea and acetic acid, while the red nanoparticles marked the F-actin filament, so that the cells were observed with red and green nanoparticles.⁸⁷ When ZnS-doped CdSe nanoparticles were bound through an amide bond into transferrin, the QDs-labelled transferrin entered cells because the receptor ion channel on cell membranes could only identify QDs-marked transferrin.⁸⁸ This result indicates that this method can be used to study the reaction or molecular exchange between donor–receptor in living cells, and also indicates the feasibility of QDs in labelling living cells, thus opening a new route for application of QDs into cells. At present, high-resolution and high sensitivity research on life activities in vivo by using QDs is a hotspot and leading edge in the field of cytobiology.^89–91

CdSe/ZnS QDs were bound with goat anti-mouse IgG or streptomycin and used as secondary antibody and anti-Her2 single clone antibodies into immune response and thereby into specific detection of breast cancer cells⁹² (Fig. 8). Fig. 9 shows the fluorescence spectrum of breast cancer cells prepared according to a report that QDs can emit fluorescence of different colors.⁹³

Fig. 8 Detection of cancer marker Her2 with QD–IgG and QD–streptavidin. Reprinted with permission from ref. 92. Copyright 2003 Nature Publishing Group.

Fig. 9 3-Color immunocytochemical staining of MCF-7 epithelial breast cancer cells. Reprinted with permission from ref. 93. Copyright 2011 American Chemical Society.

In recent years, the potent toxicity of QDs has alerted wide concern. A large proportion of materials for preparation of QDs are very toxic. For instance, Cd²⁺ will be slowly released from Cd-containing QDs, and the released amount is increased with the rise of QDs concentration, which leads to gradual enhancement of cytotoxicity and the occurrence of adverse effects.⁹⁴ Though QDs can be protected by adding a coating layer, there is little research into the long-term stability and environmental impacts of coating layers.

Biomolecular sensors are one major direction in the field of analytical biochemistry. In particular, fluorescent nano-biomolecular sensors are a novel type of sensors rapidly developing in the field of biomolecular sensing technology. Because of small volume, high sensitivity, rapid response and high anti-interference ability, fluorescent nano-biomolecular sensors are extensively applied in biology, chemistry, medicine, and other fields. So far, the ability of fluorescent nano-biomolecular sensors in detection of active substrates is still limited. Thus, it becomes a challenge for scientific workers to design novel high-performance fluorescent nano-biomolecular sensors and apply them into detection of bioactive molecules or active substances in vivo.

4. Room-temperature phosphorescence (RTP) sensors

In 2007, Thakar discovered the phosphoresce of ZnSe/ZnMnS/ZnS QDs in water solutions for the first time,⁹⁵ which facilitates the application of QDs in RTP analytical chemistry. Thakar predicted this non-toxic QDs would be extensively applied into chemical/biological analysis, and thereby created a new field of QDs biomolecular sensors. In particular, Mn-doped ZnS QDs are typical phosphorescent QDs. ZnS is a broad-band-gap semiconductor and its conduction band and valence band provide a very broad energy-level range for doping ions. Because of similarity in ionic radius and charge, Mn²⁺ can be easily doped into ZnS lattices without largely impacting the lattices. It is generally believed that Mn²⁺ emits phosphorescence in the following way.⁹⁶ Optical excitation induces electrons–holes separation in ZnS, then Mn²⁺ captures the electrons–holes and turns to the excited state, and when Mn²⁺ returns to the ground state, the energy is released in the form of phosphorescence.

Compared with fluorescence, the RTP of QDs is characterized by high sensitivity, high selectivity, low detection limit, simple synthetic steps, continuous operation, and automation. A more important characteristic is its longer lifetime. Thus, auto-fluorescence or scattering can be avoided during phosphorescent detection.^97,98 Moreover, phosphorescence is more rare than fluorescence, and thus, the selectivity can be further enhanced.^99,100 The QDs-based RTP detection does not require any deoxidant or inducer and avoids complex pretreatment.^98,101,102 In recent years, there are some achievements about the application of RTP QDs in biomolecular detection and drug analysis.^{97,98,100–109} Enoxacin which can statically quench RTP QDs is used into detection of biological fluids, and its mechanism is that the bivalent metal ions on surface of RTP QDs bind with 3-carbonyl of enoxacin to form non-phosphorescent complexes.⁸⁴ RTP QDs were also combined with methyl violet and octa(γ-aminopropyl)silsesquioxane to form nanohybrids for DNA detection.¹⁰² QDs were combined with glucose oxidase to form nanohybrids for enzyme-phosphorescent detection of glucose. A phosphorescent fingerprint sensor was developed.¹¹⁰

In general, despite the achievements made from some brand-new projects in recent years, the application of QDs phosphorescent sensors in biological detection has been rarely studied. The existing studies about biological systems are not very deep. The application in the biological field is basically at the preliminary stage, and thus abundant innovative research or refined works are needed before phosphorescent QDs become a mature technique and can be widely applied in biology.

5. QDs nanohybrids

Nanohybrids integrate the advantages of composites and nanomaterials, and more fully utilize the adjustable structural parameters and complex effect of materials, generating the optimal macro-performance. The development of nanohybrids becomes a major part of nanomaterial engineering.

Preparation of QDs nanohybrids is an efficient way to find out new properties of QDs and is very significant for development of novel biological nanomaterials. QDs can aggregate via a chemical cross-linking agent to form nanohybrids that possess higher performance than pure nanoparticles. Thus, QDs nanohybrids become a research hotspot in materialogy and are extensively applied.^{40,102,111–118} The new properties of nanohybrids are not only associated with the size and nature of compositional materials, but also with the between-nanoparticle distance and layered structure.^{40,102,111–120} Noncovalent interaction is a major method for regulation of structure and between-component distance of nanohybrids.^{102,121–126} So far, various chemical reagents including biomacromolecules, polymers and amylose can be used to form QDs nanohybrids through non-covalent interaction.^127–133 Although many methods are used in synthesis of QDs nanohybrids, it is very important to develop new biological nanohybrids for synthesis of QDs nanomaterials.

5.1. QDs bioconjugated nanohybrids

Most of the methods for assembly of QDs bioconjugates are based on protein chemical labelling, including carboxyl, amines, as well as thiol group in proteins or peptides. These functional groups interact with QDs to form cross-coupling, and ideal conjugates can be prepared after repeated purification. Under ideal conditions, these conjugates possess these properties: (1) control over the ratio of biomolecules attached per QDs, i.e. valency; (2) control over the orientation of the biomolecule attached to the QDs; (3) control over the distance separating the biomolecule from the QDs; (4) control over the strength or stability of the attachment between the QDs and biomolecule; (5) not alter or impair the function of either the QDs or biomolecule; and (6) be amenable to all manner of QDs and biomolecule. Fig. 10 briefly shows these basic principles.¹³⁴ However, the QDs nanohybrids prepared from most of these chemical methods do not possess these properties.¹³⁵

Fig. 10 Schematic of the six criteria critical to the controlled attachment of any protein to any NP or surface. In this example, the proteins would cover the NP surface in three dimensions and could still have some rotational freedom around the axis connecting them to the NP while still fulfilling the criteria. These criteria can be extended to the interaction of any biosensing molecule with the solid-state components of any biosensing device. Reprinted with permission from ref. 134. Copyright 2006 Nature Publishing Group.

5.2. QD–protein molecular imprinting nanohybrid sensors

Protein separation and identification are a basis and support for the development of proteomics. Thus, development of more effective and selective protein analytical methods becomes the key link in proteomic research. Because of high matching degree between particle sizes of QDs and biomacromolecules, and because of high spectral quality, QDs are major nanomaterials for sensing and tracing biomacromolecules.^136–138 However, since the direct adsorption of proteins by QDs is non-specific and very low selective, designing novel QDs nanohybrids probes is very necessary, which can be used into selective identification and analysis of target proteins. So far, QDs sensors based on natural antibodies are commonly used in protein identification. Because of high specificity and sensitivity, such QDs sensors have been successfully applied into medical diagnosis and environmental analysis.^21,139,140 However, the application of these sensors is restricted by the complex preparation and high cost of natural antibodies. Compared with natural antibodies, the artificial antibodies prepared through molecular imprinting are superior with simple preparation process, high mechanical performance, high identification selectivity, high chemical stability, and strong environmental tolerance. Moreover, the very high selectivity provides some simple high-performance methods for protein separation and analysis.

So far, there are already reports about the use of molecular imprinting materials, especially fluorescent printing nanohybrids, as the identification unit of novel nano-sensors.^141–146 The detection principle is that the energy transfer between QDs and template molecules will quench the fluorescence of printing nanohybrids. As reported, a pentachlorophenol phosphorescent sensor was made from printing of Mn-doped ZnS RTP QDs.¹⁴⁷ Though QDs-based printed nano-sensors are widely used into analysis and detection of small-molecules, there are few reports about their application into biomacromolecules, because the printing sensors suitable for small-molecules may not be applicable for biomacromolecules because very large volume and complex structures.^79–81

6. QDs-Based electrochemical biosensors

Electrochemical biosensors are identification units that combine electrochemical detection devices and bioactive identification materials. Because of simple manufacture process, high specificity and high sensitivity, these sensors are widely applied in biomolecular detection^148–153 and clinical medicine.^154,155

QDs possess special electrochemical and electrically-induced chemiluminescence properties, and can be used as electronic carrier in electron transfer and as photon receptor in photon absorption/conversion. The photon absorption will lead to the formation of electrons–holes very near the surface of QDs. However, electrons–holes can easily induce oxidation–reduction,¹⁵⁶ which is manifested as the sensitivity level of current response. For instance, owing to the advantages of Au nanoparticles (e.g. large specific surface area, high adsorption ability, high applicability and high electrical conductivity), DNA-functionalized Au nanoparticles and avidin-modified electrodes can be combined to build an electrochemical DNA sensor with signal magnification. With high sensitivity, high specificity and simple detection process, this sensor can be used into detection of picomole-scale target DNA and separation of single-base mismatched single-strand DNA.¹⁵⁷ Since glucose oxidase during catalytic decomposition of glucose produces H₂O₂, these authors made a QDs electrochemical sensor combining QDs and glucose oxidase for detection of glucose.¹⁵⁸ Since H₂O₂ prepared with horseradish catalase and dihydroxybenzene can quench the electrochemical luminescence of QD cathode, competitive immunoassay can be used into IgG detection¹⁵⁹ (Fig. 11).

Fig. 11 Construction (A) and incubation (B) of the immunosensor, and ECL detection without (C) and with (D and E) the enzymatic amplification by consumption of H₂O₂ as coreactant. Reprinted with permission from ref. 159. Copyright 2010 American Chemical Society.

Electrochemical immunosensors, which are a major part of electrochemical biosensors, can identify the target substances by using the electrical signals changes due to the antigen–antibody high-specific binding. These immunosensors are superior with high maneuverability, rapidness, sensitivity, low cost, easy integration and miniaturization, and thus become a research hotspot.¹⁶⁰ The use of nanomaterials further improves the sensitivity of electrochemical immunosensors.¹⁶¹

7. Summary and perspective

So far, QD-based biomolecular sensors have been widely applied into biomolecule recognition and marking and greatly promoted the development of biology and biomedicine. They are expected to play critical roles in further promoting the development of biology and biomedicine. However, there is a long way before QDs will be used into biomolecular recognition and marking in vivo. There are still some limitations. (1) Organisms or environmental samples which are compositionally very complicated often severely interfere with QDs in recognition and marking, or even lead to false positive results. Thus, one major trend regarding the development of QD-based biomolecular sensors would be how to modify the surface properties or recognizing groups of QDs, such that biomolecules have specific identifying ability like antigen–antibody. (2) One key problem regarding QD-based biomolecular sensors would be how to simultaneously identify and detect multi-component biomolecules in complex organism or environment. One major solution may be the use of biomolecular array sensors. (3) One key influence factor on the development of QD biomolecules is how to synthesize high-flux and water-soluble QDs. (4) The bottleneck limiting the clinical application of QDs is how to reduce the biotoxicity and improve the biocompatibility of QDs, especially the photoluminescent cadmium QDs. The existing research about cytotoxicity of QDs is focused on indices such as cell counting, cell morphology, apoptosis degree, and metabolic activity. The use of different indices largely complicates the between-study comparison as well as studies on the toxicity of QDs. The solution to this problem depends on research about the specific mechanism underlying the biotoxicity of QDs. The cytotoxicity of QDs is attributed to the production of more reactive oxygen species (ROS), increased permeability of lysosome, DNA damage, and gene expression alteration, all stimulated by the heavy metal ions released from the QDs.^162–164 The cytotoxicity of QDs may also be induced by the surface oxidation on QDs.¹⁶⁵ Moreover, the surface physicochemical properties of QDs are key influence factors on their toxicity.¹⁶⁶ However, the findings in this part are largely different among studies and not very systematic. Thus, deeper studies are needed to uncover the mechanism about the biotoxicity of QDs. This will be a long process of persistent explorations.

Acknowledgements

This work was supported by the funds of the Natural Science Foundation for Young Scientists of Shanxi Province (201601D021109), Shanxi Normal University (ZR1501) and School of Life Science (SUYKZ-41).

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