Chapter 1: Design of In Situ Cytosensing Strategies

Owing to the continuing attention devoted to human health and life, recent decades have witnessed tremendous developments in analytical strategies for the in situ probing of chemical and biological events at the molecular level in living cells. These tools provide spatiotemporal information on cellular molecules, biochemical reactions and signalling and communication processes,^1–3  thus contributing to the better understanding of the sophisticated mechanisms of life and to improvements in or even transformation of disease diagnosis and treatment concepts and techniques.^4,5  To analyse cellular functional molecules in situ, one needs to design a detection system composed of a recognition module, a signal transduction module and a signal output module. The recognition module achieves the differentiation of the targets by specific binding and the signal transduction module converts the recognition event into a signal, transformation or reaction to indicate the existence of the target. The latter can be integrated with the capability to amplify the signal or implement multiplexed analysis. The last module performs the final collection and reporting of the signals that correspond to the target. Owing to the low expression levels of the targets and the complex biological environment, four key aspects should be taken into consideration when designing the system: recognition/binding specificity, signal generation pathway, signal amplification and multi-channel analysis capability. These issues play crucial roles in the analytical performance and the comprehensive consideration of these aspects is challenging. This chapter presents a review of the most promising trends regarding these major concerns.

Specific detection depends on the recognition of a biosensing interface towards the target. Common recognition motifs include antibodies, lectins, peptides, nucleic acids (including aptamers), biomimetic polymers, artificial receptors and small molecules. To improve the detection sensitivity, it is beneficial to use recognition motifs with high specificity and avidity towards the target, which are often intrinsic properties of the individual recognition pairs. In spite of this, one can still tailor the detection schemes to enhance the binding intensity and specificity. For example, to improve the avidity of lectins towards their corresponding glycan epitopes, the glycans can be assembled in a multivalent fashion to accelerate the binding events. Based on this strategy, Ding et al. fabricated a mannan-modified gold electrode to yield a carbohydrate monolayer with multivalent binding capability with the lectin concanavalin A (Con A), thus permitting efficient competition between the mannan monolayer and cell-surface mannose.⁶ 

To increase the specificity for the identification of a given type of cells, Rudchenko et al. developed automata to perform cascade strand-displacement reactions directed by antibody recognition towards cell-surface markers (Figure 1.1).⁷  The automata checked the existence of each marker successively according to an “if yes then proceed” logic. Only when all the markers of the given set were present on the cell surface could the automata complete the computation and output a final fluorescence signal, thus achieving the identification of a specific population of lymphocytes within human blood cells.

Concerning the targeting delivery, most current cell targeting strategies rely on only one receptor on the cell surface and therefore usually suffer from the issue of high non-specific interactions. The incorporation of two factors in a targeting system to function in a serial manner may improve the site-specific delivery of the probe. On the basis of this concept, Ren et al. used an auto-cleavable hairpin structure acting as “smart keys” to modify the siRNA-loaded oligonucleotide nanovehicle (siRNA-ONV) and bound two kinds of aptamers, sgc8c and sgc4f, on the cell surface to act as “double locks” (Figure 1.2).⁸  These “locks” could be opened sequentially by reacting with the “key” in a serial manner. The “dual lock-and-key” mode controlled the cell “locked-open” status and thus achieved cell-subtype-specific recognition and precise probe delivery.

In addition to dual specificity control of the same type, different types of specificity control can also be integrated into one system. For intracellular sensing, considering the complex intracellular environment, it is beneficial to turn off the response capability of the recognition motif of the detection probe until it reaches the desired locations, otherwise the recognition and the subsequent signal generation may take place during the cellular delivery and uptake process, leading to impaired detection accuracy and low signal-to-noise ratio. In this context, Zhao et al. designed a nanodevice containing two dependent components: a UV light-activatable DNA aptamer probe and lanthanide-doped upconversion nanoparticles (UCNPs).⁹  The Cy3-modified aptamer strand was initially locked by a quencher-bearing complementary DNA containing a photocleavable (PC) group, which was denoted a “PC inhibitor”, hence the aptamer could not bind to ATP in its locked state and displayed a low fluorescence signal background. When the nanodevice reached the desired region, upon irradiation with near-infrared (NIR) light the PC inhibitor would be cleaved at the PC site by the UV light converted by UCNPs and the capability of the aptamer to switch its structure to bind ATP would be restored. Thus, in the presence of ATP, the dissociation of the cleaved PC inhibitor led to a substantial increase in the fluorescence signal, achieving ATP sensing with high specificity.

When the recognition motif for the target is fixed, a signal transduction pathway must be designed to indicate the occurrence of the recognition/binding events. To achieve high-sensitivity detection and to lower the background signals, a key point is the design of a signal off–on switch that can be specifically triggered by the target or target-associated events to reflect the existence of the target in the analysis scheme. The tailoring of signal generation methods depends on the action modes of the recognition processes, for example, cleavage by an enzyme or hybridization with a DNA strand.

With regard to cellular enzymes with cleaving capability, it is convenient to design signal switches by use of responsive substrates. One can label the substrate with a fluorophore at one end and use the other end to link to a nanomaterial with desired optical properties, thus constituting a certain type of energy transfer pathway, such as fluorescence resonance energy transfer (FRET). The reason for the introduction of nanomaterials lies in the synergistic employment of their unique characteristics not only from an optical properties perspective but also with regard to the accelerating capability for cellular delivery.^10,11  However, FRET-based detection platforms often suffer from a high background signal due to acceptor bleed-through. A smart solution for this issue is the design of a time-resolved fluorescence resonance energy transfer (TR-FRET) system using persistent luminescence nanoparticles (PLNPs), in which the luminescence of the fluorophore is collected only after a delay time, thus suppressing the acceptor bleed-through. Zhang et al. demonstrated this concept by developing a TR-FRET platform for the in situ lifetime quantification of intracellular caspase-3 (Figure 1.3).¹² 

Another common design for enzymes with cleaving capability is to load a peptide-based fluorophore-labelled substrate on nanomaterials with fluorescence quenching ability, such as graphene oxide (GO),¹³  gold nanorods (AuNRs),^14,15  manganese dioxide¹⁶  or a metal–organic framework (MOF).¹⁷  The cleavage by the corresponding enzymes leads to a signal-on switch by releasing the fluorophore into solution. For example, Zhang et al. fabricated a caspase-3-responsive multifunctional nanoprobe by assembling a porphyrin, a folate targeting motif and a dye-labelled peptide substrate in an MOF cage. Upon executing enhanced photodynamic therapy (PDT), owing to the integration of porphyrin and MOF, this probe allows in situ cell apoptosis monitoring by displaying caspase-3 activation-induced fluorescence recovery, thus underlining its great promise in precision cancer therapy.¹⁷ 

Cathepsin B (CaB) is another analyte that has attracted much attention owing to its crucial roles in cancer progression and diagnosis, and displays a specific peptide-cleaving capability.¹⁸  Taking CaB as the target, Tian et al. developed a multi-functional theranostic nanoplatform by the non-covalent assembly of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-{folate[poly(ethylene glycol)]-2000} (DSPE-PEG2000-FA) and chlorin e6 (Ce6)-labelled CaB-specific peptide substrate (Ce6-GRRGKGGFFFF, Ce6-Pep) on the surface of the GO sheet.¹³  Ce6, a fluorescent dye and a highly efficient photosensitizer,¹⁹  can be inhibited when in close proximity to the GO sheet. Upon folate receptor-mediated endocytosis into cells, the peptide on the GO sheet could be specifically cleaved by CaB to release the Ce6 into solution with recovery of fluorescence. This signal could be used not only for CaB imaging but also for monitoring the therapeutic efficacy under 660 nm irradiation, because the diffusion of the lighted Ce6 into cytoplasm suggested lysosomal destruction, which is a key step in the lysosomal cell-death pathway. To decrease further the background signals coming from incomplete quenching of the fluorophore by the single use of nanomaterial, Shen et al. encapsulated gold nanoparticles (AuNPs) functionalized by Cy3-labelled substrate peptide in a ZIF-8 MOF shell to achieve the synergetic use of AuNPs and MOF for superior fluorescence quenching. Here, only upon release from MOF by a lysosomal acidic environment can the peptide encounter CaB and be cleaved.²⁰ 

These signal-on design principles can also be adapted for cellular functional molecules (telomerase, miRNA, mRNA, etc.) with a specific binding capability towards oligonucleotides. In this context, the target-induced conformation or length change of the oligonucleotide substrate by hybridization (in the case of miRNA or mRNA) or elongation (in the case of telomerase) can lead to a signal off–on switch. For example, although it has been widely recognized that telomerase plays an essential role in ensuring the normal telomere length,²¹  the in situ monitoring of telomerase activity has long been regarded as a challenge. To resolve this issue, Qian et al. used telomerase primer to wrap fluorescein-loaded mesoporous silica nanoparticles (MSNs), which were also covalently modified with a black hole fluorescence quencher on their inner walls, for in situ “off–on” imaging of intracellular telomerase activity.²²  The primer could be specifically extended by telomerase and formed a rigid hairpin-like DNA structure, which could detach from the MSN surface and result in the release of originally loaded fluorescein and thus recovery of the fluorescence. In a later study by the same group,²³  this signal switch was upgraded to reduce the background signal coming from the non-specific dissociation of the primer from the MSN surface in cytoplasm by designing an AuNP-based vesicle kit. This kit contained a telomerase primer and a Cy5-tagged molecular beacon (MB)-functionalized AuNP. The hybridization of the primer extension product with the MB led to the opening of the MB and thus a signal-on switch. Although this strategy utilized a stable Au–S bond to immobilize MB on the nanoparticles, thus reducing background signals, the complex composition of the kit requires further simplification. Driven by this requirement, the group further designed a nicked MB-functionalized AuNP probe.²⁴  The telomerase-triggered elongation of the primer, which originally hybridized to the stem of the MB, led to inner chain substitution hybridization, opening of the MB and thus a signal-on switch (Figure 1.4).

With respect to miRNA as the target, Liu et al. conjugated a dye-labelled MB as the recognition substrate and signal switch, along with folic acid as the targeting motif, on AuNR. The hybridization of target miRNA with the nanoprobe led to the unfolding of MB, resulting in the off–on imaging of miRNA-21 in HeLa cells.¹⁵  Zhang et al. also employed their versatile TR-FRET platform for off–on detection of miRNA-21. The signal switch was generated by the sandwich hybridization-triggered FRET from PLNPs to fluorescein isothiocyanate (FITC).¹²  Jia et al. used a Ce6-labelled ATP aptamer to functionalize MoS₂ nanoplates and developed a nanoprobe for the fluorescence imaging of intracellular ATP.²⁵ 

Biomolecule-based signal amplification is a major type of amplification scheme that is widely used for in situ cellular analysis. Typical representatives include enzymatic catalysis and DNA nanotechnology-based strategies. For cell-surface glycan detection, Cheng et al. used horseradish peroxidase (HRP)-labelled lectin for the specific recognition of glycans on living BGC-823 cells which were captured on an electrode modified with RGDS peptide-functionalized single-walled carbon nanotubes (SWNTs).²⁶  Based on the enzymatic catalytic reaction of HRP towards the oxidation of o-phenylenediamine (o-PD) by H₂O₂, a highly sensitive cytosensor for the quantitation of cell numbers and cell-surface glycans was developed. Moreover, the biocompatible nanocomposite offered another level of signal amplification owing to the large surface area and excellent electrical conductivity of SWNTs, which contribute to both cell capture and signal amplification in electrochemical detection. This strategy was further developed to be implemented in a disposable sensor array system, allowing the detection and monitoring of multiple glycans on living cells (Figure 1.5).²⁷ 

Concerning DNA-based amplification techniques, the specificity and predictability of nucleic acid base pairing provide a rich design space for developing amplification systems.^28,29  Although several nucleic acid cascade reactions, such as polymerase chain reaction (PCR) and rolling circle amplification (RCA), have benefited the ultrasensitive biosensing of various targets, they are not suitable for in situ cellular analysis because of their dependence on exotic enzymes or high temperature. In this context, non-enzymatic catalytic amplification strategies hold great promise for improvement of the performance of in situ detection.^30,31  For example, programmable hybridization chain reaction (HCR) and catalysed hairpin assembly (CHA) have been used in intracellular microRNA,^32,33  mRNA^34,35  and telomerase imaging³⁶  for the enhancement of sensitivity.

Wu et al. designed a CHA-based signal amplification for mRNA imaging in living cells.³⁴  The DNA circuits contained two components: two hairpin-shaped metastable DNA substrates (H1 and H2), whose conformational transformations could be catalytically triggered upon target binding, and a reporter DNA duplex with the two strands respectively labelled with fluorophore and quencher. In the presence of target mRNA, a stable hybridized duplex between H1 and H2 could be generated with the target mRNA being released to catalyse the next cycle for H1–H2 duplex formation. This H1–H2 duplex could further dehybridize the reporter, leading to the restoration of the initially quenched fluorescent signal. This design permitted the generation of multiple signal outputs from one mRNA target, thus achieving the sensitive imaging of low-expression mRNA targets.

Inspired by the DNA nature of telomerase primer, Yan et al. combined the biological function of telomerase with isothermal nucleic acid-based signal amplification for the in situ detection of telomerase activity with enhanced sensitivity.³⁶  This concept was implemented through the design of a liposome nanoprobe, which consisted of a telomerase-targeting responder–transmitter DNA complex (HPT) module and a CHA signal amplification module. Upon transfection into living cells, the extension of HPT led to the release of the transmitter DNA, which could act as the initiator and catalyst of CHA amplification, resulting in the lighting up of the reporter complex. Because of the recycling use of the transmitter, multiple enhancement of signal outputs from one extension event was achieved. This intracellular CHA-based signal amplification was also integrated on the surface of UCNPs by Huo et al. to develop a nanoamplicon comparator (NAC) for miRNA imaging. The catalytic circuit responded to target miRNA and allowed quantitative UCNP-to-organic-fluorophore luminescent resonance energy transfer (LRET) imaging against a native UCNP emission reference channel.³³ 

The CHA not only can be used for signal amplification, but also provides a facile solution to reflect the protein-specific glycosylation stoichiometry by anchoring the catalytic strand on the target protein and H1 on the glycans, which allows the cyclic use of protein strands.³⁷  Similarly, exonuclease³⁸ - or restriction endonuclease³⁹ -aided recycling “hybridization and cleavage” processes between the strands respectively labelled on protein and glycans offer an alternative solution to achieve the cyclic use of a DNA probe labelled on protein.

In addition to CHA, HCR, a target-triggered alternating hybridization reaction between two hairpin probes (H1 and H2), is also widely used for signal amplification in cellular analysis. Wu et al. developed an electrostatic DNA nanoassembly format that realized intracellular HCR for ultrasensitive mRNA imaging in living cells (Figure 1.6).³⁵  This design relied on the assembly of a core AuNP, an interlayer of cysteine-terminated cationic peptides and an outer layer of fluorophore-labelled DNA probe H1. The core AuNP permitted efficient quenching of fluorophores labelled at H1 due to surface energy transfer. In the presence of target mRNA and also free-diffused H1 and H2, a chain reaction occurred by alternating hybridization between H1 (using AuNP-carried H1 as the initiator) and H2, generating a rigid double-stranded DNA structure, which could dissociate from the AuNP and activate a FRET signal indicating target mRNA expression. The multiple assembly of the H1 and H2 pair led to efficient signal amplification with a picomolar limit of detection.

Despite their powerful capability to enhance sensitivity, the kinetics of these cascade reactions depend on the diffusion of the DNA probes, which prolongs the reaction time and thus compromises the reaction efficiency. We believe that the solution to this problem lies in the confinement of DNA probes in a compact space. As an illustrative model of this concept, Ren et al. designed a DNA “nano string light” (DNSL) responsive to target mRNA based on an accelerated DNA cascade reaction (DCR) along a DNA nanowire.⁴⁰  This method achieved sensitive mRNA imaging in living cells with a highly enhanced reaction rate and efficiency. The DNSL was constructed by interval hybridization of DNA hairpin probe pairs (self-quenched H1 and H2) to a DNA nanowire with reduplicated sequence segments generated by RCA (Figure 1.7). The hybridization of the intracellular target survivin mRNA with one H1 in DNSL triggered the cascade hybridization of H1 and H2 along the DNA nanowire, which could instantly light up the whole DNSL with a highly amplified signal gain. Notably, the designed DCR could complete the reaction in a time period that is only 15% of that for traditional HCR and the amplification ratio is 20.3 times of that for HCR, demonstrating the superior efficiency of the DCR for signal amplification.

Nanoscale materials (1–200 nm) with unique optical, magnetic, electrical and photothermal properties provide ideal platforms for enhancing electronic or optical signals during in situ cellular analysis. Owing to their large surface-to-volume ratio, a wide variety of signal molecules can be loaded on/in nanomaterials, such as enzymes, DNA, redox reagents, fluorophores and other types of nanomaterials, thus contributing to the enhancement of the response coming from a single recognition event and improvement of analysis performance. To improve the detection sensitivity for cellular glycans, Ding et al. fabricated HRP and lectin co-functionalized AuNPs with a molar ratio of HRP to lectin of 8 : 1, using lectin for recognizing glycans and HRP for amplifying electrochemical signals.⁴¹  The anchoring of the nanoprobes onto electrode-captured cells led to a detection sensitivity that was 34 times higher than that using simple HRP-labelled lectin, verifying the advantages of the designed nanoprobe.

Another type of nanomaterial-based signal amplification is electrochemical stripping analysis of metal element-containing nanoprobes. In this context, the nanoprobe needs to be modified with a recognition motif and the recognition event possesses a positive or negative correlation with the target. The stripping voltammetric measurement of a recognized nanoprobe after dissolution actually brings massive signal species to the electrode surface to implement signal enhancement. As an example, Ding et al. developed a one molecule–two surface competition format for reflection of the extent of cell-surface mannose expression by the amount of lectin-functionalized quantum dots (QDs) captured by a mannan-modified electrode.⁶  An anodic stripping signal of Cd²⁺ from the dissolved product of QDs was then employed for sensitive quantification. In another example, for the detection of cell-surface sialic acid (Sia), Qian et al. used 3-aminophenylboronic acid (APBA) and avidin co-functionalized carbon nanohorn to label the aldehyde groups generated by sodium periodate oxidation of cell-surface Sia via a biotin hydrazide linker. The APBA moieties further contributed to the capture of multiple mannan-conjugated AuNPs, which could be released, dissolved and subjected to a pre-electrooxidation and then differential pulse voltammetric detection. Here, the multiple binding of AuNPs to the nanohorn probe amplified the detectable signal, thus providing a sensitive strategy for the detection of Sia and monitoring the dynamic variation on living cells.⁴² 

To maximize the detection sensitivity, researchers often integrate multiple signal amplification protocols together and design cascade signal amplification schemes. For example, Han et al. designed a double signal amplification strategy for cell-surface glycan detection: (1) developing a sandwich binding format based on the recognition of APBA-functionalized QDs towards cell-surface Sia groups and polysialic acid-stabilized AuNPs (PSA-AuNPs); and (2) making use of the sensitizing effect of cadmium cations, dissolved from QDs, on the fluorescence signal of the non-fluorescent metal-sensitive dye Rhod-5N. Thus highly sensitive fluorescent analysis of cells with a limit of detection down to eight human gastric carcinoma (BGC) cells and dynamic fluorescent monitoring of Sia expression variation on the cell surface were achieved.⁴³  This nanomaterial assembly-based signal amplification strategy can also be used for other imaging modalities. In a surface-enhanced Raman scattering (SERS) imaging strategy for cell-surface Sia,⁴⁴  a phenylboronic acid group-functionalized gold nanoflower was used as a bridge probe for both recognition of target Sia and assembly of poly(N-acetylneuraminic acid)-modified gold nanoparticles, leading to plasmonic coupling of two kinds of gold nanoprobes in a single-core–multi-satellite nanostructure. The efficient interparticle plasmonic coupling achieved could serve as a significant contributor to the enhancement of Raman signals.

The simultaneous detection of multiple targets can provide complementary information on multiple targets, thus contributing to a comprehensive understanding of the biological processes and revealing the relationships between different targets. The need to design multiplexed in situ analysis strategies is also due to the molecular characteristics shared by the large number of biological samples, hence using the expression pattern as a discriminant criterion is beneficial for precision diagnosis.

For multiplex analysis of cellular functional molecules, electrochemical sensor arrays, with the capability of location-based resolution, offer sensitive and convenient tools by integrating micromachining and nanotechnology. An excellent example of this approach is the disposable electrochemical cytosensor array developed by Cheng et al. for the simultaneous analysis of four types of glycans on intact cell surfaces.²⁷  This cytosensor could be used for monitoring of the dynamic variation of the glycan expression pattern on cancer cell surfaces during drug inducement or erythroid differentiation of human leukaemic K562 cells, suggesting its feasibility for revealing carcinoma cell-surface glycome.

The same group subsequently fabricated a microplate-based cell array to develop a chemiluminescent (CL) imaging strategy for the simultaneous detection of sialyl and galactosyl groups on cell surfaces.⁴⁵  These two types of glycans could be substituted with an aldehyde group by chemo- and enzymatic-selective oxidation, respectively, which could be lit up through aniline-catalysed hydrazone ligation with biotin hydrazide, avidin-mediated assembly of HRP and biotin co-functionalized AuNPs and CL imaging. Owing to its high sensitivity, the method achieved the distinguishing of cancer cells from normal cells and monitoring of the dynamic Sia expression on living cells.

Although location-based resolution strategies can provide multiplexed expression extents of cellular functional molecules, different signal channels collect information from cell populations at different locations. Hence these methods fail to reflect the relative quantity of information from the same cell. In this regard, label-based resolution, including spectral resolution and sequence resolution, represents a promising solution.

Spectral resolution-based multi-channel analysis can be designed for systems comprising target species of the same type or different types. For example, for members of the caspase family, which participate at different apoptosis stages/pathways using different caspase enzymes,⁴⁶  real-time revealing of the caspase evolution during cell apoptosis is essential for elucidating the cell apoptosis mechanisms. To achieve this, Zhang et al. designed a composite nanoprobe by conjugating two types of dye-labelled peptides, specific to upstream caspase-9 and downstream caspase-3, respectively, as the signal switch and folic acid as a targeting motif on an AuNR surface (Figure 1.8).¹⁴  The fluorescence of the two dyes was initially quenched by AuNR. Upon nanoprobe endocytosis and NIR irradiation, cell apoptosis was triggered by the photothermal effect and the two types of peptides could be successively cleaved by the activated caspases from upstream caspase-9 to downstream caspase-3, thus leading to the recovery of the fluorescence of the two dyes. The spectral-resolved turn-on signals permitted the quantification of both caspase-9 and caspase-3 activities in cancer cells and monitoring of their evolution in living mice and also provided an efficient handle to assess therapeutic efficiency.

As an illustration model for the multi-channel analysis of different types of targets, Liu et al. developed a black phosphorus–manganese dioxide nanoplatform for monitoring oxygen self-supply, enhancing PDT and feeding back the therapeutic effect (Figure 1.9).¹⁶  The hybridized nanoplatform was prepared by assembly of rhodamine B (RhB)-encapsulated manganese dioxide (R-MnO₂) as O₂ supplier and indicator and FITC-labelled peptide-functionalized black phosphorus as the theranostic agent. After being delivered into cancer cells, the nanocomposite dissociated in the acidic and H₂O₂-rich environment and generated O₂ to overcome hypoxia-associated PDT resistance. The amount of O₂ released showed a proportional relationship to the liberation of Mn²⁺ and RhB, hence the fluorescence of RhB could be used as an indicator of O₂ self-supply. Upon PDT, the cleavage of peptide by the activated caspase-3 in apoptotic cells occurred, leading to the recovery of the fluorescence of FITC and the achievement of real-time feedback of the therapeutic effect. In addition to PDT, the system featured a dual-monitoring capability, which provided cues for selecting the laser treatment timing and optimizing the therapeutic dose.

Although using different dyes to label different targets is a common approach to achieving the imaging of multiple targets in living cells, it becomes intricate when it comes to visualization of multiple glycans on a given protein at the same time. One needs to label simultaneously both the protein and the target glycans with fluorescence donors and acceptors to design FRET-based imaging systems to report the proximity of the protein and its carried glycans. It should be noted that the number of dyes doubles as the number of glycan target channels increases. However, owing to the difficulty of obtaining appropriate pairs of donors and acceptors without interference and the serious acceptor bleed-through resulting from the discrepancy between protein copy number and glycan abundance,⁴⁷  it is not feasible to report the protein-specific glycan expression by multiplexed FRET.

To solve the problem, inspired by the unique polychromatic light-emitting property of UCNPs, Wu et al. used UCNPs as a protein-confined single donor to construct a duplexed luminescence resonance energy transfer system (D-LRET) to activate simultaneously multiple acceptors labelled on glycans via single near-infrared excitation.⁴⁸  Upon 980 nm excitation, the UCNPs exhibited two energy emission bands, each of which matched and lit up one kind of fluorescent dye in close proximity (within 10 nm) through LRET, allowing the visualization of distinct glycoforms of mucin 1 on various cell types and also quantitation and monitoring of the relative expression ratio of termini glycans (Sia and fucose) on mucin 1.

Despite the effectiveness of the above system for imaging two types of glycans on a specific protein, this strategy cannot mirror the authentic hierarchical structure of DNA, RNA or proteins, all of which have extrinsic, single- or multi-level appendage structures emanating from the respective core structures, because relying on spectral resolution will inevitably limit the number of available signalling channels. To meet the challenge, we proposed to encode an arbitrarily expandable number of structural motifs in these biomacromolecules with distinct identification codes. In this regard, the DNA barcoding technique that makes use of the sequence diversity of DNA for identification of different targets provides a useful handle. In addition to the abundant barcodes achievable, it also offers another valuable advantage, namely smooth connection of these codes with various DNA amplification/detection techniques.

As a proof-of-concept demonstration, Li et al. developed a hierarchical coding (HieCo) imaging strategy, with DNA coding and decoding of protein and monosaccharides executed in fidelity to the hierarchical order of the target glycoprotein, for mucin 1-specific imaging of terminal Sia and fucose on tumour cells (Figure 1.10).³⁷  The protein and monosaccharide codes were initially masked and the decoding process began with the timing code and proceeded in a sequential, iterative decoding–unmasking fashion. Specifically, the protein code could decode different monosaccharide codes in a catalytic cycle manner, thus allowing the relative quantification of the extents of expression of different monosaccharides. Distinct monosaccharide patterns for four types of cells were observed and the dynamic monitoring of mucin 1-specific monosaccharide pattern changes associated with both the alteration of cellular physiological states and the occurrence of epithelial-to-mesenchymal transition was achieved. This approach featured expandable monosaccharide identification channels and was regarded as “an important step in the development of a system analogous to the use of green fluorescent proteins for protein tagging” by Suzuki.⁴⁹ 

This chapter summarizes the design concepts for the in situ analysis of cellular functional molecules and provides insightful solutions for addressing the challenges in this field. In spite of these advances, further development of in situ cytosensing methods is still urgently needed, with numerous opportunities available. For instance, future work will be focused on developing novel nanoprobes by precision synthesis and functionalization. This may endow probes with homogeneous surface properties in addition to site-specific and quantity-predetermined assembly of recognition/signal motifs, and thus will contribute to the decrease of the background signal and enhancement of detection sensitivity. The combined introduction of enzyme-, light- or pH-responsive functional units along with the recognition module into the detection system provides another dimension to control the timing and region of the recognition events, which will facilitate precision detection. To fit the wide dynamic range of some functional molecules, which is due to the biological heterogeneity, it is beneficial to develop multiplexed analysis methods in a multimodal way to combine the inherent strengths of each modality to maximize sensitivity, specificity, spatial resolution, temporal resolution, signal penetration, etc., and obtain extensive complementary information on the targets.