Controlled Release Of Cargo

Targeted cargo delivery, as described in previous section, can be combined with remotely controlled cargo release. Remote activation is based on an external physical stimulus such as light, ultrasound, or radio frequency, which acts on colloidal nanoparticles present in the walls of the capsules and leads to rupturing/permeation of the walls of the capsules so that the cargo is released from the cavity to the environment. Irradiation of noble metal and magnetic particles with light and radio frequency waves, respectively, leads to local heating as described earlier and subsequent disintegration of the capsule walls. Alternatively, sonication of capsules functionalized with colloidal nanoparticles in their walls leads to mechanical disintegration of the capsule walls. In this way, the cargo can be released from the cavity of the capsules following an external trigger. Ultrasound and radio frequency fields can penetrate tissue well, but they are complicated to focus. For this reason they are favorable for cargo release from capsules deep inside tissue. Visible light, on the other hand, is strongly absorbed by tissue but can be easily focused to micrometer size spots. For this reason, controlled opening of individual capsules inside single cells and subsequent release of their cargo to the cytosol is possible (fig 1) .Short irradiation (<10> not ,very similar35 mW) is sufficient for opening capsules without damaging cells and tissues. While moderate irradiation disintegrates the walls of capsules, releasing molecules from their cavity, higher doses of irradiation can increase the local temperature so far as to destroy surrounding cells, an effect known as hyperthermia. Local accumulation of heatable capsules (i.e. with noble metal and magnetic nanoparticles in their walls which are responsive to light and radio frequency exposure, respectively) in cancerous tissues could be used for induced heating mediated by the nanoparticles involving subsequent tumor deterioration. As only the temperature of cells close to the particles is raised, the heated cells would be selectively killed, without exposing the entire organism to elevated temperatures.

Fig. 1. Controlled-release of cargo with light-responsive capsules. (i) Laser irradiation of noble metal (e.g. Au) nanoparticle-functionalized capsules leads to (ii) local heating of the metal NPs, and (iii) subsequent rupture of the capsule wall.

In summary, nanoparticle functionalized capsules offer the potential to combine all of the above listed concepts into one single system for drug delivery (fig 2). Magnetic nanoparticles in the capsule walls allow direction and accumulation of capsules at the designated target region upon application of external magnetic field gradients. Ligands immobilized on the capsule surface, which are specific for receptor molecules present on target cells, would permit specific and enhanced cellular uptake via receptor-ligand binding. Fluorescent semiconductor nanoparticles in the capsule walls allow monitoring of the transportation and uptake process using fluorescence microscopy. Capsules inside target cells could be opened with a laser pointer by heating gold nanoparticles in the capsule walls which then releases the cargo molecules. Combination of these methods is expected to lead to greater specificity in drug delivery. This procedure could be a great advantage to anticancer therapy, as selective toxicity in tumor cells could be achieved via local chemotherapeutic damage. Nevertheless, to be applicable in clinical practice, several additional hurdles will have to be overcome. First of all, to reach a specific tissue, intravenous administration is often required. Therefore, the capsules should be limited in size as they could obstruct the smallest blood capillaries. From the circulation point of view, the upper limit for an ideal vehicle size should not exceed 200 nm. Secondly, due to their polyionic nature, the capsules tend towards protein adsorption, potentially leading to capsule aggregation in the blood capillaries.

Fig. 2. Target delivery systems. (a) Geometry of polymer capsules loaded with magnetic (black), fluorescent (green), and gold (yellow) NPs in their walls. Ligand molecules are attached to their surface and filled with cargo molecules (grey star-shape). (b) Schematization of targeted local cargo delivery into cells with multifunctional polymer capsules. Capsules allow for delivery to target cells by local accumulation with magnetic field gradients and specific binding to receptors on the target cells, and for controlled release of cargo inside cells upon light-controlled opening of capsules.

Besides the potential use for targeted and controlled release of drugs, cargo delivery with capsules can be seen in the more general concept of triggered local delivery. Caged Ca has been used for decades in biology, in the local and controlled delivery of Ca ions. For this purpose, Ca is introduced into chelating complexes from which it can be released by light-illumination. Noble-metal functionalized capsules could serve the same purpose, although they would allow for light stimulated release of macromolecules from the cavity of the capsules. Stimulated local release of enzymes and DNA would be of particular relevance. Release of enzymes could, for example, trigger the conversion of prodrugs within cells into active drugs. Also short oligonucleotide sequences, such as si-RNA, could be introduced as cargo inside the capsule cavities for gene delivery applications. In particular, such capsules could provide an easy way to improve the current transfection methods used in molecular biology as they would lead to more efficient translational arrest of specific transcripts from RNA inside living cells. In this role, the incorporation of single-stranded antisense oligonucleotides, linear double-stranded and plasmid DNA into polymer nanospheres has been demonstrated.

Polyelectrolyte Capsules As Carrier Systems For Local Sensing

Sensing based on optical measurements is very attractive as it allows the transduction of chemical concentration information into optical signals which can be quantified. Fluorescent indicators are a class of fluorophores whose spectral properties are sensitive to a substance (the analyte) of interest. Numerous indicators are commercially available for a variety of analytes, including Ca²⁺, Mg²⁺, Cl^-, H⁺, Na⁺, and O₂. Such analyte-sensitive fluorophores can be integrated into polymer capsules. Due to the size-dependent permeability of the walls of the polymer capsules, analyte-sensitive fluorophores of high molecular weight can be maintained in their cavity, whereas analyte molecules of low molecular weight can diffuse in and out freely. A microcapsule-based pH-sensor system using the seminaphthorhodafluor dye (SNARF-1) has already been described (fig 8a). Whereas capsules in the alkaline cell medium are fluorescent in the red, capsules which have been incorporated into cells inside acidic compartments are fluorescent in the green (fig 8a). Such pH-sensitive capsules present an interesting tool for high throughput quantification of cellular uptake. Besides the fact that naturally incorporated capsules are trapped inside intracellular compartments and thus are not in contact with the cytosol – a problem which will be discussed later – there are several advantages to embedding analyte-sensitive fluorophores into capsules. Firstly, long term measurements could be achieved, as the fluorophores inside the capsules are protected against enzymatic degradation and the cell is protected from the free fluorophore. Secondly, as many fluorophores are embedded in each capsule, there is a high local fluorophore concentration, which enhances their sensitivity (fig 8c).

Fig. 8. Capsule-based pH sensor. (a) Capsules are loaded with the pH-sensitive fluorophore SNARF in the cavity, which fluoresces green in acidic and red in alkaline environments. (b) SNARF-molecules inside capsules change their color from red in the extracellular alkaline environment of a culture medium to green upon internalization of a capsule into acidic intracellular compartments. (c) When the same number of SNARF molecules is either microinjected into a cell or introduced into a capsule carrier, the SNARF is diluted over the whole cytosol in the first case or locally concentrated inside the capsule in the second case. Local concentration of the SNARF leads to higher signal-to-noise ratios.

Most importantly, in contrast to alternative technologies such as the probes encapsulated by biologically localized embedding (PEBBLE) system, capsule-based sensors do, in principle, allow for multiplexed measurements. This is based on the fact that capsules can be functionalized with fluorescent molecules at two distinct positions, in their walls and in their cavities. In this approach, fluorophores sensitive to different analytes are loaded into the cavities of different capsules and the walls of each capsule are fluorescently labeled with a barcode (fig 9a). The color of the capsule wall would allow for identification of each capsule and thus provide the information for which analyte this particular capsule is sensitive. The local analyte concentration could be derived upon recording the fluorescence resulting from the cavity. As fluorophores for sensing different analytes are locally separated by embedding them in different capsules, spectral overlap between the different fluorophores is no longer a problem. Presumably, this concept would allow for the detection of multiple analytes in parallel in the cytosol of single cells (Fig 9b).

Fig. 9. Multiplexed sensing. (a) Capsules with semiconductor NPs as barcodes embedded in their walls and analyte-sensitive fluorophores inside their cavities. The fluorescence of the wall (here shown with blue, green, or red fluorescent NPs) is used to identify which type of analyte-sensitive fluorophore is loaded in the capsule cavity and thus to which analyte the sensor responds. (b) By loading different sensor capsules into one cell, different analytes can be detected in parallel.

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Fig. 9. Multiplexed sensing. (a) Capsules with semiconductor NPs as barcodes embedded in their walls and analyte-sensitive fluorophores inside their cavities. The fluorescence of the wall (here shown with blue, green, or red fluorescent NPs) is used to identify which type of analyte-sensitive fluorophore is loaded in the capsule cavity and thus to which analyte the sensor responds. (b) By loading different sensor capsules into one cell, different analytes can be detected in parallel.

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However, as mentioned, the delivery of capsules to the cytosol remains a fundamental problem for multiplexed measurement of different analytes inside cells. Though capsules can be directly introduced to the cytosol by electroporation, delivery to the cytosol via active capsule incorporation by cells would be preferable. However, as mentioned in earlier, capsules taken up by cells are stored inside intracellular compartments. One way of direct delivery to the cytosol might be the modification of the capsule surface with virus-derived ligand molecules, such as TAT-peptides which have been successfully used for the delivery of nanoparticles into cells. An alternative strategy is presented in fig 10. By repeated coprecipitation, double-shell capsules can be synthesized. The inner capsule could now be a sensor capsule with a fluorescent barcode in the walls and analyte-sensitive fluorophores in the cavity, while the wall of the outer capsule could be functionalized with noble metal nanoparticles. Light-illumination of such double-shell capsules inside intracellular compartments would lead to local heat generation in the metal particles. Therefore, it can be speculated that the heat would be sufficient to rupture and permeate the outer wall of the capsules as well as the membrane of the vesicular compartment in which the double-shell capsule is trapped. In this way, the intact inner sensor capsule would be released into the cytosol. Although this concept still has to be proved experimentally, there are experimental data that support the idea. Indeed, light-illumination of single metal particle-functionalized capsules not only permeates the capsule walls, but also the surrounding membrane of incorporated capsules, as demonstrated upon release of fluorescent cargo from the capsule cavity to the cytosol. Although vesicular membranes around the capsules must have been locally disintegrated, cells have been demonstrated to tolerate this treatment.

Fig. 10. Release of capsules into the cytosol. Double-shell capsules could act as a sensor capsule with a fluorescent barcode in the inner capsule wall and analyte sensitive fluorophores in the central cavity, and noble metal NPs in the outer capsule wall. Illumination of the capsules would cause heating of the metal NPs followed by disintegration of the outer wall and release of the intact inner capsule.

In Conclusion

Multifunctional polyelectrolyte capsules fabricated by the LbL assembly technique possess remarkable properties, even though they are held together by electrostatic attraction. In particular, this very general assembly mechanism allows for the integration of virtually all different types of charged nanoscale objects into and on top of their walls. Introduction of nanoscale objects introduces functionality (such as fluorophores, magnetic particles, and local heat sources) and specificity (such as biomolecular ligands). The cavity of the capsules can be filled with cargo, which is to be released at designated targets or with active molecules, as for example for multiplexed sensing. Compared with other carrier systems, capsules can be functionalized at two distinct compartments, walls and cavities, which introduces flexibility for the interference-free introduction of multiple functionalities.

It has to be clearly pointed out that the individual concepts outlined in this article for use with capsules have previously been suggested for other types of carrier systems. Magnetic drug targeting, receptor-ligand based targeting, hyperthermia, and photo-induced release of cargo are concepts well known in medical research. The novelty of the capsules as a carrier system lies in the possibility of combining all these different strategies into one single object and thus provide true multifunctionality.

Most of the concepts mentioned in this article have been experimentally demonstrated on cell cultures and the experimental data can be found in the references. In particular, a proof of principle for a microcapsule-based system for biospecific target delivery and for local analytic detection in small volumes in cell cultures has been successfully realized by several groups. Though several groups have started with experiments on animals, enormous hurdles still have to be overcome before applying polymer capsules to clinical practice. Of particular importance will be the prevention of capsule aggregation in blood vessels, the control of capsule clearance by the organism, and the synthesis of highly biocompatible capsules.

Novel Nanostructures For SERS Biosensing

Surface-enhanced Raman scattering (SERS) is a powerful analytical tool for chemical and biological sensing applications. However, one feature which has limited its use in biosensing applications is the difficulty involved in producing uniform, highly sensitive, and reproducible SERS substrates. Recent developments in oblique angle deposition and other nanofabrication techniques have overcome this limitation, providing an unprecedented opportunity to develop SERS substrates for pathogen biosensor applications. Recently reported examples of SERS's newfound sensing abilities include the capacity to detect low levels of viruses and bacteria, as well as to discriminate between types and strains of pathogens, including pathogens with gene deletions. A brief review of our recent progress in SERS biosensing is given in this article.

Article Outline

SERS Detection dictated by substrate fabrication

Pathogen Detection

Distinguishing between different virus types

Detecting Viruses In Biological media

Detecting Viruses Captured Onto The Nanostructures SERS Surface

SERS Detection of Low Levels Of Virus

Detecting different Strains Of A Single Virus Type

The rapid and sensitive detection of pathogens is critical for disease intervention strategies, as well as the control and prevention of pandemics and acts of bioterrorism. There is a need to be able to perform pathogen detection, both in laboratory facilities, and under field conditions. Consequently, biosensing platform technologies are under development to allow for both applications. Current methods of virus and bacteria detection generally employ antibody-based assays such as enzyme-linked immunosorbant assays (ELISA), fluorescent antibody assays, or serologic evaluation for exposure. Many of these assay methods provide only a limited level of sensitivity, thus low level pathogen detection generally requires nucleic acid amplification coupled with polymerase chain reaction (PCR) assays.

More recently, other diagnostic methods such as microcantilevers, evanescent wave biosensors, immunosorbant electron microscopy, and atomic force microscopy have been investigated to overcome some of the limitations of sensitivity, but these new techniques are unable to effectively discriminate between types and/or species of pathogen with reasonable sample throughput. The research directions for improvement of biosensing methods require reduction or elimination of sample preparation or amplification procedures. Detection and discrimination of specimens in complex biological media are also a necessity, together with reproducible results, cost and time effectiveness, and ease of use under most conditions.

SERS Detection Dictated By Substrate Fabrication

Surface-enhanced Raman scattering (SERS) has emerged as a powerful analytical tool that extends the possibilities of vibrational spectroscopy to solve a vast array of chemical and biochemical problems. SERS is an extension and variation of standard Raman spectroscopy, a vibrational spectroscopic technique that provides detailed information about the materials under investigation at the molecular level. Since the discovery of the SERS effect in the 1970s, SERS has been applied to a wide variety of analytical applications, including biochemistry and the life sciences. As has been reviewed in detail elsewhere, two primary mechanisms are believed to be responsible for SERS enhancement: a long-range classical electromagnetic (EM) effect and a short-range chemical (CHEM) effect. These two mechanisms contribute simultaneously to the overall enhancement; EM is thought to contribute the most (10–10) to the observed intensity enhancement, while CHEM is thought to contribute a lesser amount (10–10).

Since SERS is useful for determining molecular structural information and also provides ultrasensitive detection limits, including single molecule sensitivity, it has been used to detect pathogens that include bacteria and viruses. There are two principle SERS configurations that have been used in biosensing, intrinsic or extrinsic, as shown in fig 1. In intrinsic detection (Fig 1a), the analyte can be directly applied to the nanostructured surfaces and the inherent Raman spectrum of the biomolecule directly measured to identify the specimen. To allow for capture and to aid specificity of detection, antibodies, aptamers, or related molecules can be immobilized onto nanostructured surfaces as shown in fig 1b and the Raman spectral differences before and after capture of the specimen can be used to identify the species. In extrinsic detection, a Raman reporter molecule is used to generate a signal for detection. For example, a Au nanoparticle may be used as the SERS-active substrate to which a Raman reporter molecule is immobilized (fig 1c ). By coating this structure with another layer of dielectrics such as SiO₂, TiO₂, or a polymer, a core-shell complex is formed in which the outer-shell may be decorated with capture molecules such as antibodies. Thus, specimens may be captured and detected via a sandwich structure as shown in fig 1c . This extrinsic SERS detection method has been successfully used for in vivo SERS imaging of unique or rare cancer cells.

Fig. 1. Different SERS detection configurations: (a) Direct intrinsic detection; (b) indirect intrinsic detection; and (c) extrinsic detection.

The remarkable analytical sensitivity of SERS has yet to be translated into the development of widely accepted, commercially viable diagnostic applications, due in large part to the difficulty in preparing robust, metal-coated substrates of the correct surface morphology that provide maximum SERS enhancements. Some of the important requirements for an ideal SERS substrate in practical diagnostic applications are that the substrate produces a high enhancement, generates a reproducible and uniform response, has a stable shelf-life, and is simple to fabricate.

Many substrate preparation techniques exist that can form roughened metal surfaces of the types required for ideal SERS enhancements. These methods include roughening of a surface by oxidation-reduction cycles (ORC), metal colloid hydrosols, laser ablation of metals by high-power laser pulses, chemical etching, roughened films prepared by Tollen's reagent, photodeposited Ag films on TiO₂, and vapor-deposited Ag metal films. While the majority of substrate preparation techniques reported to date focus on the problem of achieving large SERS enhancements, the other requirements listed above for the production of a practical SERS sensing substrate are seldom addressed. Currently, there are five fabrication techniques that could potentially produce the desired SERS substrates to meet these requirements: electron beam lithography, nanosphere lithography, the template method, the hybrid method, and an oblique angle vapor deposition method.

The electron beam lithography (EBL) method is an ideal method for producing uniform and reproducible SERS substrates. Unfortunately, it is very expensive to produce large area substrates using EBL, unless the technique is combined with a nanoimprint lithography method. The nanosphere lithography (NSL) method pioneered by Van Duyne and coworkers involves evaporating Ag onto preformed arrays of nanopore masks by colloid particles, which are subsequently removed, leaving behind the Ag metal deposited in the interstices to form a regular Ag nanoparticle array. The template method utilizes a nanotube-like array, such as anodized Al₂O₃, as a template to deposit Ag or Au nanorods directly into the channels via an electrochemical plating method. Hybrid methods fabricate SERS substrates by depositing metal particles onto nanoporous scaffolds such as porous silicon, nanorod arrays, etc. The oblique angle deposition (OAD) method is based on a conventional physical vapor deposition principle and can be used to fabricate aligned and tilted Ag nanorod arrays on large substrate areas. For OAD fabrication, the surface normal of the substrate in a vacuum chamber is positioned at a very large angle with respect to the incoming vapor direction (> 75^o), as shown in fig 2a This deposition configuration results in a so-called geometric shadowing effect that leads to a preferential growth of nanorods on the substrate in the direction of deposition. The nanorods grow aligned but tilted on the substrate as shown in fig 2b. The benefits and limitations of the different methods for preparation of SERS-active substrates are summarized in table 1

Fig. 2. (a) Schematic of oblique angle deposition. (b) Top view, and (c) cross-sectional view scanning electron micrographs of the Ag nanorod array SERS substrates.

Table 1.

Comparison of different SERS substrate fabrication techniques that could potentially generate uniform and large area SERS substrates

Fabrication Method	EBL	NSL	Template method	Hybrid Method	OAD
Enhancement Factor	–	107–10 9	106–10 7 (50,52,53)	106–10 8 (59)	>108 (64)
Substrate Area (cm2)	Typically <>	1 x 1	> 2.5 x 2.5	> 2.5 x 5.0	> 2.5 x 7.5
Uniformity (%)	–	–	<15>	–	<>
Reproducibility	<20>	–	–	–	<>
Shelf time (days)	–	–	–	> 40 (for Au)	7 (for Ag)
Fabrication Steps	3	3	3	> 2	1–2
Cost	Expensive	Inexpensive	Inexpensive	Inexpensive	Moderate

Pathogen Detection

Several research groups have used these new nanofabrication methods to produce highly sensitive and reproducible SERS substrates for biosensing applications. Studies have been conducted to evaluate SERS quantitatively as a biosensing method for pathogens such as viruses and bacteria. Compared with some of the other pathogen detection techniques previously noted, SERS offers several advantages, including high sensitivity, type and species classification, the discrimination of subtle structural differences, and detection of differences in the nucleic acid profiles between species. We have recently demonstrated that Ag nanorod arrays, produced by oblique angle vapor deposition, offer several advantages for SERS pathogen biosensing applications. While the majority of our applications have been in the area of virus biosensing, we have also demonstrated similar capabilities for the detection of bacteria, as well as other agents including microRNAs, using these Ag nanorod arrays.

Distinguishing Between Different Virus Types

Using the direct intrinsic SERS configuration as shown in fig 1a, we have shown that it is possible to rapidly and sensitively distinguish different viruses by their SERS spectra (fig 3). For example, the baseline-corrected SERS spectra of the DNA virus, adenovirus (adeno), and RNA viruses, i.e., rhinovirus (rhino) and human immunodeficiency (HIV) viruses, which can readily be detected and distinguished as shown in fig 3. The adeno SERS spectrum is characterized by strong bands due to nucleic acid bases at 650 cm^-1 (guanine), 731 cm^-1 (adenine), 1325 cm^-1 (adenine), and 1248 cm^-1 (guanine). The 650 cm^-1 band may also have contributions due to Tyr. The Raman lines at 1003 cm^-1 and 1033 cm^-1 have been assigned to the symmetric ring breathing mode and the in-plane C-H bending mode of Phe (phenylalanine), respectively while the bands at 1457 cm^-1, 1576 cm^-1 and 1655 cm^-1 can be attributed to the CH₂ deformation mode of proteins, the carboxylate stretching vibration (v_a COO^-) of Trp (Tryptophan) and the amide I vibration of peptide groups, respectively. A notable characteristic of the adeno SERS spectrum is the relative intensity of the bands associated with the nucleic acids, indicating direct binding to the Ag substrate. The strong band at 731 cm^-1 has been assigned to denatured DNA, caused by its interaction with the Ag SERS substrate. A similar band analysis can identify the prominent SERS bands for the other viruses in fig 3. Thus, the uniqueness of the SERS spectrum provides a molecular fingerprint for detection of specific viruses and provides the foundation for SERS-based biosensing

Fig. 3. SERS spectra of different virus types obtained using Ag nanorod substrates.

Detecting Viruses In Biological Media

For most diagnostic situations, the detection of pathogens occurs in a heterogeneous biological medium. Therefore, it is of practical importance that SERS can distinguish between viruses in the presence of a complex background. This capability has been demonstrated by comparing the SERS spectra of uninfected Vero cell lysate, respiratory syncytial virus (RSV) infected cell lysate, and purified RSV (fig 4). As shown in fig 4, although there are common SERS peaks for the three samples, both the SERS spectra of RSV-infected cell lysate and purified RSV have SERS bands at 1000–1100 cm^-1 and 500–600 cm^-1, while the SERS spectrum of Vero cell lysate does not have these two signature peaks. The bands at 527 cm^-1 and 546 cm^-1 can be assigned to a disulfide stretching mode, while the strong band at 1044 cm^-1 has been assigned to the C–N stretching vibration in previous SERS studies. The results show that major Raman bands can be assigned to different constituents of the cell lysate and the virus, such as nucleic acids, proteins, protein secondary structure units and amino acid residues present in the side chains and the backbone. However, our most significant result is the observation that vibrational modes due to the virus can be unambiguously identified in the SERS spectrum of the Vero cell lysate after infection.

Fig. 4. SERS spectra of Vero cell lysate before and after infection with RSV, top and middle spectra, respectively. The SERS spectrum of purified RSV is shown at the bottom for comparison.

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Detecting Viruses Captured Onto The Nanostructured SERS Surface

The indirect intrinsic detection configuration shown in fig 1b can also be used to detect viruses captured by antibodies to increase the detection selectivity. Antibodies can be readily immobilized onto the SERS substrate, and the SERS spectra before and after virus treatment can be obtained to compare Raman signatures of antibody alone versus antibody plus captured virus. The SERS spectra of Ag nanorods coated with the IgG antibodies and viruses captured by these antibodies are shown in fig 5. Of the spectral features apparent in the IgG2a antibody spectrum (fig 5, top), the most intense band at not, vert, similar 1000 cm^-1 most likely arises from the in-plane ring deformation mode of Phe in IgG. Prominent bands are observed in the 1400–1600 cm^-1 region of the RSV and IgG complex spectrum (fig 5, bottom). This is presumably due to selectively enhanced nucleic acid and/or side-chain vibrations, although the amide III protein mode at 1260 cm^-1 may be observed in both the IgG and RSV+IgG spectra. These results show that antibodies or related capture moieties can be used to provide selectivity to SERS biosensing

Fig. 5. SERS spectra of (top) IgG2a antibody complex on Ag nanorod array, (bottom) RSV-IgG2a-Ag nanorod complex.

SERS Detecton Of Low Levels Of Virus

The sensitivity and dynamic range of the SERS technique for virus detection has been investigated by analyzing dilutions of a respiratory syncytial virus (RSV) mutant lacking the G (attachment) gene (ΔG). The SERS peak areas of the main band at 1045 cm^-1 (C–N stretching mode) are plotted against the ΔG RSV concentration in fig 6. The concentrations of the diluted solutions are calculated from the volume of water used for the dilutions. The SERS intensity increases with concentration of the viral solution, reaching a plateau at concentrations above 10 PFU/ml (PFU = plaque-forming unit). This behavior is not uncommon and similar findings of decreasing signal with increasing concentration have been reported for SERS substrates with an adsorbate coverage ≤ 0.01 monolayer. Although at this stage little emphasis have been placed on determining the lowest detectable titer, values as low as 100 PFU/mL are readily detectable. These data suggest a limit of virus detection ranging from 1–10 PFU of virus in this assay format.

Fig. 6. The SERS calibration curve for ΔG RSV constructed with the peak area or the C–N stretching band at 1045 cm-1.

Detecting Different Strains Of A Single Virus Type

In addition to the capacity to differentiate between different virus types, SERS can also be used to distinguish strains of a single virus type. One example is the detection of RSV strains (fig 7), although we have also shown that SERS can readily distinguish influenza A strains. Using SERS, the RSV viruses A/Long, A2 and ΔG (belonging to the RSV A strain), as well as the RSV strain B1, have been analyzed and their corresponding baseline corrected spectra (1400 cm^-1–600 cm^-1) are shown in fig 7.

Fig. 7. Summed SERS spectra of individual RSV strains (a) strain A/Long, (b) strain B1, (c), strain A2 with a G gene deletion (ΔG), and (d) strain A2.

The SERS spectrum of A/Long (fig 7a) differs from the other RSV spectra in that the prominent C–N stretch occurs at 1055 cm^-1, compared with 1042–1045 cm^-1 for the other RSV viruses. Bands unique to A/Long are also observed at 877 cm^-1 and 663 cm^-1, while the band at 528 cm^-1 (present in the other spectra) is absent. It is likely that the different spectrum observed for A/Long relates to a different composition of nucleic acids and viral envelope proteins. As predicted, there are also differences in the SERS spectra between the A and B1 strains. The differences that distinguished the A strain from the B strain SERS spectra include the relative intensities of the nucleic acid bands compared with the other bands in the spectrum. Significantly, we have also been able to show that the intrinsic SERS spectra are capable of detecting gene deletions in viruses. This is shown by the SERS spectra comparing the parental A2 strain (fig 7d) to the RSV G protein gene deletion mutant from which it was derived, ΔG (fig 7c). Comparison of the spectra reveals subtle yet real differences in peak intensities of the Raman spectra between 700 –900 cm^-1.

Perspectives

SERS has long been considered as a potential biosensing technology due to its inherently high sensitivity and its ability to provide unique spectroscopic fingerprints of the target analyte. Our recent studies show that, with the appropriate substrate, SERS offers a potent biosensing platform with many advantages over current biosensing or detection applications.

Despite the potential power of SERS as a biosensing tool, there remain critical and practical issues that need to be addressed before the technique can be routinely applied. One consideration is the need to produce inexpensive and reliable SERS substrates having uniformly high enhancements. Another important consideration is the reproducibility of the spectral response from the target biomolecule, which ultimately relates to the statistical reliability of the method. A practical SERS substrate fabrication method would produce an inexpensive, uniform, reproducible, and reusable SERS-active substrate. Since SERS enhancements critically depend on substrate nanomorphology, a practical fabrication method should have the ability to produce nanostructured arrays with specific size, shape, alignment, and architecture within very tight tolerances. Thus, the challenges for a practical SERS nanostructure fabrication method are the ability to:

(i) control the size, aspect ratio, and shape of nanostructures;

(ii) grow the desired nanostructure at low temperature and onto a particular substrate geometry, e.g. flat, cylindrical, or tapered;

(iii) fabricate metallic and dielectric nanostructures in a multilayered fashion; and

(iv) integrate the fabrication process with other conventional microfabrication techniques.

When considering ultrasensitive SERS detection, e.g. low viral or bacterial loads in a clinical sample, the issue of statistical sampling arises. In such cases, it is possible that when the amount of SERS analyte presents a severe limitation, surface coverage relative to the laser spot size may become an issue in detection. However, this situation may be overcome by employing analyte capture methods, e.g. using antibodies, or by concentrating the limited analyte to be detected. Solutions for these critical issues are addressable and are being investigated so that the door may be opened to a new era of biodetection.

Self-Assembling Peptide Nanotubes

Biological proteins and peptides have the intrinsic ability to self-assemble into elongated solid nanofibrils, which may give rise to amyloid diseases or inspire applications ranging from tissue engineering to nanoelectronics. Proteinaceous fibrils are extensively studied and well understood, to the extent that detailed theoretical models have been proposed that explain and predict their behavior. Another intriguing state of protein-like self-assembly is that of nanotubes (NTs), defined here as an elongated nano-object with a definite inner hole. In contrast to proteinaceous fibrils, nanotubes are much less frequently observed and far less well understood. However, they have attracted research interest internationally as key components for nanotechnology.

Article Outline

Peptide Nanotubes

Dipeptides

Linear Peptidea

Chemically Modified Linear Peptides

Cyclic Peptides

Outlook

Peptide Nanotubes

Nanotubular structures can form from a variety of different materials such as inorganic, carbon, biological microtubules, porins, viral proteins, α-lactalbumin, amyloid proteins, DNA, lipids carbohydrates, synthetic polymers, and other organic systems. Molecular self-assembly is the main bottom-up approach for the affordable production of bulk quantities of well-defined nanostructures. Biological building blocks such as DNA, lipids, and viruses are extensively studied for this purpose.

Proteins and peptides are the most versatile natural molecular bricks, due to their extensive chemical, conformational and functional diversity. They also offer specificity of interactions, necessary for biosensing, catalytic and molecular recognition processes, and scalable production either through chemical synthesis or genetic engineering. Due to the high complexity of proteins, there is a difficulty associated with the thorough understanding of the physical and chemical principles that underpin and control their self-assembling properties.

Simple model systems and short peptides offer a much more viable route to gaining a quantitative and systematic insight into protein-like self-assembly. Here we review the emerging field of self-assembling nanotubes made of simple peptide building blocks, and discuss their morphologies, applications and the future outlook. We start with the simplest systems and work towards increasingly complex systems.

Dipeptides

The simplest peptide building blocks for the construction of NTs are dipeptides from the diphenylalanine motif of the Alzheimer's β-amyloid peptide. When this peptide is dissolved at 100 mg/ml in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and then diluted down with water at a final concentration of ≤ 2 mg/ml, multiwall NTs are formed, with a typical diameter of 80–300 nm and micron length. A similar but more rigid diphenylglycine peptide analogue forms remarkably stable spheres 10–100 nm in diameter under the same solution conditions. Spherical particles also form instead of NTs when a thiol is introduced into the original diphenylalanine peptide. The occurance of either tubes or spheres provides an insight into the possible mechanism of their formation (fig 1a ), suggesting that the formation of either tubular or closed cages by fundamentally similar peptides is consistent with the closure of a two-dimensional layer, as described both for carbon and inorganic nanotubes and their corresponding buckminsterfullerene and fullerene-like structures.

Fig. 1. (a) Schematic of the formation of tubular (single or multiwalled), spherical, or fibrillar structures via dipeptide self-assembly. (b) Proposed model for the formation of aligned peptide nanotube arrays. (i) Scanning electron micrograph of the vertically aligned peptide nanotubes. (ii) Cold field-emission gun high-resolution scanning electron (CFEG-HRSEM) micrograph of the nanotube arrays. (iii) High-magnification micrograph of an individual nanotube obtained by CFEG-HRSEM.

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The NTs are stable under extreme conditions, e.g. in autoclave (121°C, 1.2 atm); circular dichroism spectra do not change from room temperature up to 90°C; dry tubes heated to 150°C are stable, while degradation occurs at 200°C. The NTs display remarkable chemical stability in a wide range of organic solvents and pH. Indentation atomic force microscopy experiments on the mechanical properties of dried NTs on mica give an estimated averaged point stiffness of 160 N/m and a high Young's modulus of 19 GPa (27 GPa in another study). This makes them amongst the stiffest known biological materials. For example, biological microtubules which provide a rigid cytoskeleton to the cell have a Young's modulus of 1 GPa. However, the stiffness of the dipeptide NTs is lower than that of carbon and inorganic nanotubes. It has been suggested that as well as intermolecular hydrogen bonding, the rigid aromatic side chains may also be responsible for stability and mechanical strength, as well as providing directionality for formation through specific π–π interactions. Biological systems are notorious for their instability and sensitivity to temperature and chemical treatments. The significant thermal and chemical stability of these NTs points to their possible use in micro- and nanoelectromechanics (MEMS and NEMS) and functional nanodevices.

Carbon nanotubes (CNTs) are extensively studied for sensing applications due to their mechanical stability, conductance and large surface area. A drawback of CNTs is the effect exposure to humidity, oxygen, N₂O, and NH₃ has on their electric properties. CNTs are also believed to pose problems in device fabrication due to lack of uniformity, hydrophobicity and thus, limited solubility, reproducibility of precise structural properties, cost, and limited opportunities for covalent modification. Therefore, the dipeptide NTs offer an attractive alternative for device fabrication. Several steps have been taken in this direction. In one example, peptide NTs have been deposited on graphite electrodes. Improved electrode sensitivity is observed, suggesting that this may be due to an increase in the functional electrode surface in the presence of NTs. In a related study, thiol-modified peptide NTs have been immobilized and dried on Au electrode surfaces and an enzyme coating was applied to them. The resulting electrodes show improved sensitivity and reproducibility for the detection of glucose and ethanol, short detection time, large current density, and comparatively high stability. The findings show that novel electrochemical biosensing platforms may be fabricated based on biocompatible NTs.

Cationic dipeptides NH₂–Phe–Phe–NH₂ self-assemble into NTs at neutral pH and rearrange into spherical structures (possibly vesicles) 100 nm in diameter, upon dilution below 8 mg/ml (fig 2). The tubes can be absorbed by cells through endocytosis upon spontaneous conversion into vesicles. This property has been used to deliver oligonucleotides into the interior of the cells as a proof of concept of the potential applications of the system in gene and drug delivery.

Fig. 2. Proposed model for the transition of DNA-loaded cationic dipeptide nanotubes (CDPNTs) into vesicles and their cellular uptake for oligonucleotide delivery.

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Dipeptide NTs have also been used to fabricate 20 nm Ag nanowires, effectively acting as a degradable casting mould. Ag ions are reduced to metallic Ag in the lumen of the tube, and the peptide template removed by enzyme degradation. This approach may have applications in molecular electronics as such small nanowires cannot be made by conventional lithography. In another related study, AgNO₃ is reduced in the hollow pores of the NTs, then thiol-containing linker peptides are bound on the outside of the nanotubes. These attached Au nanoparticles, which act as nucleation sites during the electroless deposition of a gold, cover the NTs. Thus the fabrication of metal-insulator-metal trilayer coaxial nanocables, which may give rise to unique electromagnetic properties, has been demonstrated.

In many applications, nanotubes cannot be applied as individual nanostructures, rather their macroscopic organization is necessary or preferred. Using a simple method, two-dimensional ordered films of NTs 1 μm in thickness have been created, comprising closely arranged spherulites of multiple NT bundles. This is achieved by dissolving the NTs in the carbon-nanotube-debundling solvent N-methyl-2-pyrrolidone (NMP), followed by deposition of the peptide solution on a surface (SiO₂, Au, Pd, alumina, mica, quartz, InP), heating at 60°C for solvent evaporation, and cooling to room temperature. Successful preparation of a Ag-embedded NT composite network has also been carried out using a similar approach. The authors intend to expand these experiments to form other hybrid films, thus offering opportunities for the construction of new complex nanostructures, for use in biosensors and biocoating materials.

Impressive macroscopic alignment (fig 1b ) has also been demonstrated by nanoforests of vertically aligned NTs, formed by unidirectional growth of dense arrays. This process involves application of the dipeptide dissolved in HFIP onto siliconized glass followed by solvent evaporation. It has been proposed that evaporation results in a super saturation state that facilitates the formation of numerous nucleation sites on the surface. This may then be followed by unidirectional growth of nanotubes as more peptides join the growing ends of the tubes. A method for horizontal alignment of NTs has also been demonstrated through non-covalent coating of the tubes with magnetite nanoparticles and the application of an external magnetic field. These results demonstrate the ability to form a two-dimensional dense array of these NTs with either vertical or horizontal patterns.

Linear Peptides

Next up in molecular complexity are longer designed peptide surfactants approximately 2–3 nm in length, with a hydrophilic head of one or two charged amino acids and a hydrophobic tail of four or more consecutive hydrophobic ones, e.g. A₆D, V₆D, G₈DD, KV₆. Upon dissolution in water at 4–5 mM, these peptides form a network of cationic or anionic open-ended nanotubes ( fig 3 ) with 30–50 nm diameters and numerous three-way junctions that connect them together; vesicles can fuse or bud out of the nanotubes. The 4–5 nm thick wall is formed by a bilayer of peptides. These tubes are stabilized by hydrophobic effects and they are reminiscent of the nano- and microtubes formed by lipids, but an order of magnitude smaller in diameter. However, unlike conventional surfactants, peptide surfactants pack by hydrogen bonding interactions. In fact, some peptide surfactants display typical β-sheet structure implying a fairly extended backbone. pH is also an important factor since the charge needs to be maintained in the headgoups, to avoid conversion of NTs into large membranous aggregates. Peptide purity is also found to be crucial for the reproducible behavior of peptide NTs.

Fig. 3. (a) Self-assembling nanotubes and vesicles of negatively and positively charged surfactant peptides: (i) V₆D nanotubes; (ii) V₆D vesicles budding out of nanotubes; and (iii) K₂V₆ nanotubes. Color code: green-hydrophobic tails, red-aspartic acid, blue-lysine. (b) Quick-freeze/deep-etch transmission electron micrographs of (i) surfactant peptide nanotubes; (ii) vesicles possibly budding from the nanotubes or vice/versa.

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Alanine (A) and valine (V) produce more homogeneous and stable NTs than glycine (G), isoleucine (I), and leucine (L). For cationic peptides, lysine (K) or histidine (H) are preferred over arginine (R), possibly due to steric effects. Glycine and aspartic acid (D) are also interesting because these amino acids are believed to have been present in the prebiotic environment of the early Earth. If peptides consisting of a combination of these amino acids can form nanotubes and vesicles, they would have the potential to provide primitive enclosures facilitating catalysis and prebiotic molecular evolution. It is also possible to design molecular recognition elements into the peptide, in order to deliver a wide range of substances inside the cell in a site-specific manner. Envisaged applications include carriers for the encapsulation and delivery of a number of small, water-insoluble molecules and large biological molecules, including negatively charged nucleic acids inside the cell, and cosmetic applications.

A biological peptide derived from the amphiphilic core Aβ(16-22) of the Alz peptide can also assemble into parallel β-sheets that produce bilayer structures at low pH (fig 4a). These stack on top of each other in large numbers to give rise to helical ribbons which fuse at the edges to produce highly homogeneous nanotubes (fig 4b) with a 52 nm outer diameter, a 4 nm-thick wall and lengths of several microns. The tubes can be densely coated with negatively charged colloidal Au particles to prevent further NT association. Extensive bundling (diameter of 1 μm and >5 mm contour length) of NTs can be achieved by salting out (fig 4c and 4d). Counter ion mediation allows lateral alignment of the NTs during this process. This is a simple and generic strategy to produce higher order assemblies of homogeneous NTs.

Fig. 4. Proposed model for self-assembly of Aβ(16–22) peptide nanotubes. (a) A flat rectangular bilayer stacks to form a laminate of bilayers. (b) The laminate of bilayers twist to form nanotubes. Cryo-etch high-resolution SEM micrographs of peptide nanotubes: (c) before, and (d) after sulphate bundling.

Chemically Modified Linear Peptides

Nonbiological modifications of linear peptides can help tip the balance towards the formation of hollow nanotubes as opposed to solid nanofibrils. Three notable examples illustrate this point. The first example comes from the 20–29 segment of amylin protein which undergoes amyloid β-sheet fibrillization. Several derivatives of this segment have been produced with a modified backbone to prevent intermolecular hydrogen bonding and thus act as β-sheet breakers, and abolish fibril formation. Unexpectedly, two of these derivatives form helical ribbons and NTs 200–300 nm in diameter and several microns in length. The increased hydrophobicity of these derivatives, as opposed to intermolecular backbone hydrogen bonding, is believed to be the driving force for self-assembly in this case.

In the second example, studies focused on a model peptide show the formation of solid β-sheet fibrils. Variant biotinylated peptides designed with different linkers between biotin and the peptide termini are shown by fourier transform infrared (FTIR) to adopt β-sheet structure and to form homogeneous tubes in water with an external diameter of 60 nm and an internal diameter of 30 nm. The antibiotin antibody effectively binds to biotin groups on the NTs. The authors propose using these tubes as scaffolds for proteins. In a recent study, short peptide derivatives (N-(fluorenyl-9-methoxycarbonyl) commonly known as Fmoc, Fmoc-Leu₂ and Fmoc-Leu₃, have also been shown to form gels in water consisting of nanotubes with external and internal diameters of 15.2–19.5 nm and 4–6 nm, respectively. Interestingly, they were twisted into two-dimensional β-sheets by enzymatically triggered self-assembly.

A class of well-studied NT-forming molecules containing both peptidic and non-peptidic segments are the bola-amphiphilic peptides (fig 5a), which over the course of several days in water, form NTs (fig 5b) with a wide range of diameters from 20 nm to 1 μm. In order to control their diameter, NTs have been assembled within polycarbonate membranes. Depending on pore size, the membrane can act as a template to produce NTs with a controlled width from 50 nm to 1 μm. This opens up the opportunity to apply NTs as templates for nanowires. Nanowires are essential building blocks for electronics and sensors; production of nanowires of uniform diameter is crucial for these applications as their electric and magnetic properties are sensitive to size. In one study, NTs have been immobilized on self-assembling monolayer (SAM)-functionalized Au substrates via hydrogen bonding and then metallized by nickel. This approach may be useful for nanoelecronic fabrication since the NTs can be coated with various metals to form metallic nanowires.

Fig. 5. (a) Chemical structure of the bola-amphiphilic peptide monomer, bis-(N-R-amido-glycylglycine)-1,7-heptane dicarboxylate. (b) Proposed model for the formation of peptide nanotubes. (c) Schematic illustrating the formation of lipase-loaded peptide nanotubes and their enzymatic application.

NTs have also been functionalized with proteins, nanocrystals, and metalloporphyrin coatings via hydrogen bonding. The application of these coated tubes to nanoscale, highly sensitive chemical sensors, electronics or photonics may be possible. In another study, a model lipase enzyme is encapsulated in NTs by incubation for a week (fig 5c). The catalytic activity of the nanotube-bound lipase increases by 33% as compared with free-standing lipases at room temperature. At elevated temperatures of 65°C, the lipase activity inside the NTs is 70% higher than free standing lipases. These amazing results are believed to stem from the fact that the bound lipase activity is most likely induced by the conformational change of lipases to an enzymatically active structure during adsorption.

This class of NTs can also be organized macroscopically, e.g. Ni⁺² ions employed as a bridge between free peptide amines result in bundles (diameter 100 μm and length 3 mm) of NTs. Addition of EDTA to the suspension causes disassembly of the NT bundles. Bundles will have greater mechanical stability than individual ones, which may be necessary for applications. They are also easier to handle than individual NTs. Another important property for application development is the ability to immobilize NTs at a specific location on a substrate. Accurate localization of NTs can be demonstrated by assembling antibody-functionalized NTs at specific locations on substrates (fig 6) where their complementary antigen proteins are patterned.

Fig. 6. Atomic force microscopy of antihuman-IgG-coated bolaamphiphilic peptide nanotubes attached to trenches filled with human IgG (a) before incubation with antibody-coated nanotubes – 0%, (b) when the concentration of human IgG is 70% with 30% of BSA spacer on the trench. (c) When the concentration of human IgG in the trench is reduced by 50%, antibody-nanotube attachment improves dramatically. (d) The trend continues even at a reduced concentration of 10% human IgG. (e) Maximum nanotube attachment is achieved when the concentration of human IgG in the trench is 7%, and (f) at human IgG concentrations < bars="500">