Vertically Aligned Carbon Nanotubes as a Unique Material for Biomedical Applications

Abstract

Vertically aligned carbon nanotubes (VACNTs), a unique classification of CNT, highly oriented and normal to the respective substrate, have been heavily researched over the last two decades. Unlike randomly oriented CNT, VACNTs have demonstrated numerous advantages making it an extremely desirable nanomaterial for many biomedical applications. These advantages include better spatial uniformity, increased surface area, greater susceptibility to functionalization, improved electrocatalytic activity, faster electron transfer, higher resolution in sensing, and more. This Review discusses VACNT and its utilization in biomedical applications particularly for sensing, biomolecule filtration systems, cell stimulation, regenerative medicine, drug delivery, and bacteria inhibition. Furthermore, comparisons are made between VACNT and its traditionally nonaligned, randomly oriented counterpart. Thus, we aim to provide a better understanding of VACNT and its potential applications within the community and encourage its utilization in the future.

Graphical Abstract

1. INTRODUCTION

1.1. History and Background of CNT and VACNT.

The unique properties of carbon nanotubes (CNTs) have made them well researched since their discovery by Iijima in 1991.¹ A CNT is a 2D graphene layer rolled up into a nanometer-sized cylinder. Like graphene, CNTs are composed of carbon atoms bonded via hybrid sp² bonds to three adjacent carbon atoms, forming a hexagonal structure. However, different from the flat sheet of graphene, the curvature in CNT results in some sp² bonds within the carbon network obtaining characteristics similar to a sp³ bond.^2–4 These new characteristics lead to a difference in bonding force constant in the CNT circumferential direction, making the nanotubes stronger along their axis.^5,6 Furthermore, the presence of only a limited number of carbon atoms along the CNTs’ circumference results in quantum confinement and associated π bonds that establish exceptional innate electrical properties. While not discussed here, there are other factors that also influence CNT electronic properties, namely, chirality.⁷ CNTs made of a single-rolled graphene layer, are called single-wall carbon nanotubes (SWCNTs) and have a diameter of approximately 1 nm. Whereas, CNTs made of a roll of stacked graphene (graphite), resulting in multiple concentric nanotubes, are called multi wall carbon nanotubes (MWCNTs) and can reach diameter up to 100 nm. Both types of CNT are capable of very long lengths (millimeter) making them high aspect ratio structures. Despite the structure, size, and synthesis differences, both materials generally behave similarly in biomedical applications. Thus, this Review will discuss both MWCNTs and SWCNTs.

While the properties of CNTs are vast, the lack of oriented spatial arrangement does not make them conducive for many experiments. Thus, shortly after Iijima’s initial report, advancements were made toward aligned CNT. It was not until 1996 when Li et al. initiated CNT growth by chemical vapor deposition (CVD) that CNT self-organization would truly be realized.⁸ With this method, however, something even more extraordinary came about: not only were the CNTs aligned, but they were aligned perpendicular to the substrate, establishing a new classification of CNTs referred to as vertically aligned carbon nanotubes (VACNTs). Over the last two decades, VACNTs have shown many unique characteristics and advantages compared with their nonaligned, randomly oriented counterparts. In this Review, the synthesis of VACNTs will be briefly discussed, followed by their advantages over traditional, randomly aligned CNTs. Afterward, a detailed discussion regarding the biomedical applications of VACNT will be presented. Here, research from the past two decades is culminated regarding VACNT integration in platforms for biosensing, biomolecule filtration, cell stimulation, tissue engineering, and bacteria inhibition. The aim of this Review is to present findings that support VACNT supremacy in the biomedical field and propel future research.

1.2. Synthesis of VACNT.

VACNT is primarily synthesized through two techniques: arc discharge and chemical vapor deposition (CVD). Arc discharge of CNT converts an amorphous carbon precursor into structural tubes. This is accomplished by using two electrodes: an anode filled with the carbon precursor and a cathode, typically a pure graphite rod. A direct current arc is attained between the electrodes resulting in plasma and carbon vapor drift toward the cathode. Upon reaching the cathode, the carbon is cooled and the resultant product is CNT.^9,10 To achieve vertical alignment, the electric field between the cathode and anode must be vertical such that the force induces growth normal to the substrate.^11,12

Despite producing lower crystallinity than arc discharge, CVD is more popular and conventionally used to grow VACNT since it is superior in terms of cost, yield, purity, structural control, and architecture.^10,13,14 There are three key components in CVD-based VACNT synthesis: a hydrocarbon, a catalyst, and a catalyst support. Hydrocarbon vapor passes through a high temperature (600–1200 °C) tubular reactor, where molten catalyst material is present. At this sufficiently high temperature, the hydrocarbon thermally decomposes and VACNTs grow on the catalyst in the reactor. Extensive studies have been performed to understand the carbon-catalyst interaction and VACNT growth mechanism. In 1971, Baker et al. proposed volume diffusion-based growth. Here, the hydrocarbon decomposes and establishes a temperature gradient across the volume of the catalyst nanoparticle. Carbon then diffuses down the catalyst nanoparticle due to this temperature gradient and initiates the VACNT growth from the base of the nanoparticle.¹⁵ This model is now known as the “tip-growth model” since the VACNT grows continuously from its tip. However, if the catalyst–substrate interaction is strong enough, VACNT precipitation fails to push the metal particle up and instead emerges out of the top surface of the metal. This is known as “base-growth model” since VACNT grows continuously from its base.¹³ In 2004, because of the advancements in the in situ technologies, Helveg et al. studied carbon fiber growth in real time. Their observations suggested a surface diffusion growth mechanism. As catalyst nanoparticles form in high temperature, carbon binds to the step edges, initiating VACNT growth on the surface.¹⁶ Additional in situ studies suggest VACNT nucleation is initiated by the carbon cap formation. First a graphene embryo is formed, bound between opposite step-edges of the catalyst nanoparticle. Then, the embryo grows, eventually leading to curved carbon cap formation and generating VACNT walls.¹⁷

Various types of CVD have been developed in an effort to tune VACNT physical characteristics (diameter, number of cocentric tubes, length, etc.). These methods vary in temperature, carbon precursor, environmental conditions, and more. Among all of the developed CVD methods, plasma enhanced CVD (PECVD) is the most reliable and controlled way to synthesize VACNT.¹⁰ This method is similar to the previously discussed CVD but instead uses high energy plasma to decompose the carbon precursor. Because of this, one of the main advantages of PECVD is a higher selectivity and tunability, provided by controlling the amount of ionized carbon species supplied to the catalyst. Moreover, it also offers high alignment controllability by controlling the electric field.^18,19 By using PECVD, tunable chirality and growth rate of CNT have been demonstrated, further highlighting its controllability.^20,21

1.3. Properties of CNT and VACNT.

1.3.1. General Properties of CNT.

As mentioned, the CNT’s chemical structure enables many unique mechanical and electrical properties. Specifically, the sp² hybridized orbital brings about extremely strong bonds giving CNT its high tensile strength (>100 GPa).⁶ This is stronger than diamond (5.19 GPa), which has weaker bonds from its sp³ orbitals.²² These bonds are also very stiff and efficiently transmit lattice vibrations, making CNT thermally conductive (up to 6600 W/mK at 20 °C).^7,23 Furthermore, the sp² orbitals in CNT give rise to a single π bond and, consequently, a delocalized electron, enabling conductivity as high as 10⁷ S/m.²⁴ Interestingly, the conductivity is largely dependent on the orientation of the carbon sheet, known as chirality. While not discussed here, it can make CNT either metallic or semiconducting.⁷

The rolled-up structure constitutes a hollow nanometer-sized center within the CNT.^25–27 The porosity of the structure is taken advantage of in many applications, including drug delivery and will be explored in a future section. In addition, the structure has a very high aspect ratio (as large as 10⁶:1) which is critical for many applications, such as tissue engineering.²⁸ Finally, the bonding structure of CNT makes it chemically inert and very stable.^5,6 However, the biocompatibility is less understood and extremely dependent on various CNT properties.²⁹

CNT type (single-walled or multiwalled) affects biocompatibility and toxicity, and can induce different biological responses.³⁰ Moreover, CNT impurities, such as residual metal catalyst, can affect biocompatibility, as reported by Murray et al.³¹ CNT administration and dosage also directly influence biocompatibility. For in vitro applications, mainly with diagnostics and sensing, these properties are less critical. However, for many therapeutic and drug delivery applications, understanding the role of these CNT properties is essential. Intravenous administration in rats has demonstrated that low doses of CNT can persist in many organs, including the kidney, heart, lungs, and brain without affecting acute or long-term health.³² However, some tissues, such as mesothelial tissue, are extremely sensitive to intraperitoneal administration and results in toxic effects. Additionally, CNT dosage provides a direct effect on biocompatibility and toxicity with an increase in dosage negatively affecting biocompatibility.³³ As discussed in the next section, CNT biocompatibility can be improved via functionalization and is frequently used throughout tissue engineering.^34–37 Finally, recent research suggests CNTs’ potential for degradation, thus contributing to their biocompatibility. Studies have indicated that with the correct characteristics, CNTs can be degraded, usually via macrophages.^38–41 This research provides evidence to support in vivo applications of CNT, which could lead to advances in therapies such as gene transfection.

1.3.2. Advantages of VACNT.

VACNTs have the same innate properties as randomly oriented CNTs, but their normal orientation brings about synthesis, chemical, mechanical, and electrical advantages, as outlined in Figure 1.

Figure 1.

General properties of CNT and overview of VACNT advantages, specifically regarding synthesis, chemical, mechanical, and electrical properties. VACNTs have numerous synthesis advantages including being easily patternable, in situ growth, and large effective surface area. Mechanically, VACNTs can deflect under pressure, their tips are readily accessible, and its uniform spacing and dense packing allow mimicking of extracellular matrix. Electrically, VACNT tips are more electrochemically active with higher electrotransfer rate and improved conductivity compared with randomly oriented CNT. Chemically, VACNTs have better chemical functionalization with heavy localization at the tips, high purity via in situ synthesis, and controlled wettability including superhydrophobicity and superhydrophilicity.

1.3.2.1. Synthesis.

VACNTs are more easily patterned than their counterparts.^42–44 While standard lithography can be utilized to pattern the catalyst, and therefore VACNT features, randomly oriented CNTs require complicated methods with tedious controllability. To begin, the main method for patterning CNT is using dispersions in liquid mediums. To effectively and stably disperse CNT, the van der Waals attraction forces between CNTs must be overcome to separate CNT bundles into individually dispersed CNT. Contacting CNT have energy per contact length of approximately 30–40 kT nm⁻¹, so energy greater than this must be supplied to break the aggregation.⁴⁴ However, due to their large aspect ratio and interaction volume, Brownian motion inevitably leads to more aggregation; thus individually dispersed CNTs are difficult to achieve. Nevertheless, there are a multitude of methods used to nanoscale pattern CNTs from dispersions, such as dielectrophoresis (DEP) or substrate modification, but they are tedious, require specialized equipment, and are low-throughput.⁴⁴ Furthermore, these methods attach CNT horizontally on the substrate and can even result in CNT overlap, both of which limit the effective CNT surface area to volume ratio.⁴⁴ Lower surface area to volume ratio consequently decreases the effectiveness of many CNT properties, such as surface functionalization and electrocatalytic activity.^42,45–48 This, however, does not occur in the case for VACNT since the normal direction of growth naturally yields a higher surface area to volume ratio and the van der Waal forces during synthesis prevent entanglement of VACNT.^13,44,49 In fact, Brownlee et al. calculates that VACNT has an effective surface area 15 times larger than randomly oriented CNT.⁴²

1.3.2.2. Mechanical.

The vertical aspect ratio enables many unique applications of VACNT.^26,43 While their tensile strength is still extremely high, VACNTs can deflect under pressure (Poisson’s ratio of 0.19 and Young’s modulus of 1–10 MPa).^50–54 This mechanism is dependent on the length of the VACNT: deflection angle increases with increased height. As discussed later, this makes them great bactericide and antibiofilm platforms. Furthermore, the normal orientation makes VACNT tips readily accessible. These nanometer-sized tips can pierce cellular membranes or proteins for enhanced chemical monitoring or malignant cell mutilation.^55–58 Finally, the uniform spatial arrangement and lack of overlapping, as described earlier, of VACNTs promote densely packed structures^47,59 and better match the extracellular matrix⁶⁰ of the human body so they can act as scaffolds for tissue engineering.

1.3.2.3. Electrical.

VACNT tips have been shown to have faster electron transfer rate than randomly oriented CNT.^{45,46,48,61,62} This is largely because of the chemical structure of the CNT. Because of its rolled structure, the tips of the VACNT act similar to the edge planes of pyrolytic graphite, while the walls act like the basal planes.^63,64 Graphene edge planes have higher electron transfer than basal planes, thus the tips have higher transfer than the walls. In fact, Fayazfar et al. directly compared the electrocatalytic activity of randomly oriented CNT to VACNT via cyclic voltammetry and found it to be greater in VACNT.⁴⁵ This, coupled with the greater surface area and uniformity, make them excellent electrodes. Moreover, VACNTs were shown to generate electric fields with low potentials, which can be utilized for inducing cell polarization.⁶⁵ For these reasons, compared to randomly oriented CNT, VACNT are widely used in many electrochemical sensor applications.

1.3.2.4. Chemical.

CNT tips have more defects than their sidewalls, making VACNT very susceptible to functionalization.^{35–37,61,66,67} This has been validated using near-infrared spectroscopy and Raman spectroscopy, specifically on the tips of VACNT, which shows the effectiveness of chemical modification.^66,68 This is critical in many electrochemical sensors and biosensors, where antibodies are functionalized to immobilize target molecules.^45,46,64,69 Moreover, oxygen-plasma functionalization has seen great success in creating carboxyl groups at the tips of VACNT.⁶⁷ It is theorized that the defects might favor the formation of graphene oxide and hence favor the formation of polar groups when treated with oxygen plasma.³⁵ This modification changes the superhydrophobic nature of VACNT to superhydrophilic, which is critical for cellular adhesion in tissue engineering. Finally, the CVD synthesis method of VACNT leaves little metallic and amorphous carbon residue due to the high reaction temperature and high residue removing efficiency by the hydrogen and carrier gas.⁷⁰ This is critical for biocompatibility, and thus, the pristine condition of in situ-grown VACNT is necessary for many therapeutic applications.

1.3.3. Disadvantages of VACNT.

Despite the numerous advantages of VACNT, there are inherit disadvantages which limit its usage. To begin, VACNTs, by definition, must be oriented and attached normal to a substrate. This constraint limits their usage in applications such as drug delivery or gene therapy which traditional CNTs have capability. While both VACNT and CNT can store nanomolecules within their hollow center, VACNTs cannot deliver the cargo in vivo since they are bound to a substrate. These traditional CNT applications are outside the scope of this Review, but have been discussed in detail elsewhere.^27,71 During VACNT synthesis, the van der Waal forces between the tubes is needed to support growth, thus achieving a single VACNT is not possible unlike traditional CNT.^13,44,49 This, coupled with the confinement to a substrate, prevent VACNT usage in applications, such as electronic nanowires.⁷² Another limitation is the need for specialized equipment to synthesize VACNT. The most common method, thin film catalyst patterning followed by CVD, require photolithography, catalyst deposition, and a reaction furnace.^10,13,14 This, coupled with the material costs, makes VACNT synthesis an expensive endeavor. Once synthesized, the VACNTs are also susceptible to delamination. While the tensile strength is high, the average delamination energy is approximately 0.6 pJ/CNT.⁷³ While this does allow VACNT transfer onto new substrates, enabling a variety of applications, it also presents device fabrication challenges. Finally, commercialization bottlenecks have severely limited the clinical uses of VACNT. This will be discussed further in the conclusion of this Review.

2. BIOMEDICAL APPLICATIONS

Nanomaterial-integrated devices offer many benefits over traditional devices such as small sample size, lower cost per analysis, and point-of-care capability. Specifically, VACNTs have become increasingly researched because of their advantageous properties listed earlier. In this section, different VACNT systems, including biosensors and biomolecule enrichment devices, will be presented. Then, other applications of VACNTs will be reviewed in regards to cell stimulation and recording, drug delivery systems, cell scaffolding for tissue engineering, and antibacteria platforms.

2.1. Biosensing.

2.1.1. Biosensors for Proteins.

Conventionally, proteins are detected by enzyme-linked immunosorbent assay (ELISA). This assay utilizes an antibody–antigen binding affinity to capture the target molecule, then an enzyme-linker is added, which, if the target was captured, sends a signal to the ELISA plate reader.⁷⁴ Typically, the linear dynamic range for ELISA is limited to approximately 2 logs, while the specificity and sensitivity depend on the specific capture antibody.⁷⁵ A major limitation of ELISA, however, is the lack of real-time, continuous monitoring. Because, typically, the target binding can not be undone, the assay quickly becomes saturated with already occupied binding sites. Thus, targets can not be detected continuously. Finally, because the ELISA plate reader measures via a spectrophotometer it inherently struggles with light absorbance issues and background interference. For these reasons, protein sensors utilizing VACNT have been heavily investigated, many of which have achieved remarkable performance.

The first CNT-integrated protein sensor dates back to 1997.⁷⁶ Here, the conductive nanosurface of CNT acted as an electrical contact to monitor the electrochemical response of protein cytochrome c.⁷⁶ Nevertheless, because of the random orientation of the CNTs, the spatial relationship between the proteins and the nanotubes was inconsistent and reduced protein contact to the electrode, making the system less effective. In contrast, however, VACNTs are more densely packed and have enhanced electron transfer between the electrode surface and the redox label.⁷⁷ It was not until 2003 when Gooding et al. took advantage of this and used vertically aligned single walled carbon nanotubes (SW-VACNT) to improve the electrochemical monitoring of proteins. Here, they describe that the alignment of SW-VACNTs improves communication with the redox proteins since they spatially match their redox center more uniformly.⁷⁸ Soon after, Yu et al. developed a similar platform but severed the ends of the SW-VACNT to selectively localize carboxylate groups to improve functionalization of antibodies.^61,79 As listed in Table 1, this shortened CNT was used in conjunction with an enzyme to detect low levels of HSA,⁶¹ PSA,⁸⁰ MMP-3,⁸¹ and IL-6^82,83 via amperometry measurements. More recently, another enzymatic SW-VACNT sensor was developed for the sensitive detection of carcinoembryonic antigen (CEA).⁴⁸ However, instead of the reduction of a peroxidase inducing a current, a glucoamylase enzyme catalyzes the hydrolysis of starch to release glucose. This glucose then reduces an Au salt attached to the nanoprobe releasing Au nanoparticles which are detected via differential pulse stripping voltammetry (DPSV) (Figure 2A). This innovative method allowed for detection limit to reach as low as 0.48 pg/mL (Table 1), which is well beyond that of ELISA.⁴⁸

Table 1.

Novel VACNT-Based Biosensors for Various Target Molecules

General Target	Target Molecule	Sensing Method	LOD	Sensitivity	Electromaterial	Ref.
protein	human serum albumin (HSA)	Antibody & Enzymatic	1 pmol/mL	46 nA/pmol·mL	Shortened SW VACNT	61
	prostate specific antigen (PSA)		4 pg/mL	1.12 μA/μM·cm2		80
	matrix-metalloproteinase-3 (MMP-3)		4 pg/mL	77.6 nA/log		81
	interleukin-6 (IL-6)		0.5 pg/mL	19.3 nA mL/pg·cm2		82,83
	carcinoembryonic antigen (CEA)		0.48 pg/mL	Not reported.	SW VACNT & Au Salt	48
	prostate specific antigen (T-PSA)	Antibody & Impedance	0.25 ng/mL	Not reported.	SW VACNT	84
	human cancerous inhibitor PP2A (CIP2A)		0.24 pg/mL	Not reported.	MW VACNT	85
	biotin		1 ng/mL	Not reported.		42
	carcinoembryonic antigen (CEA)	Antibody & Redox	0.5 pg/mL	0.052 μA/(ng/mL)		86
	thrombin	Antibody on Field Effect Transistor	Not reported.	33 μA/μM·mm2	SWCNT gated MOSFET	87
Cell	colon cancer cells	Impedance	4000 cells/cm2	1.7×10−3 Ω cm2	MW VACNT	58,88
	renal Carcinoma cells (RCCs)	Cell Traction Forces	Single Cell	Not reported.		50
	lung cancer cells (QUDB)	Folate Receptor	Not reported.	4×10−3 Ω cm2		89
	leukemia cells (K562)	Antibody & Impedance	10 cells/mL	0.506 μA/(cell/mL)		90
nucleic acid	DNA (hybridization)	ssDNA Probe & Impedance	Not reported.	Not reported.	Shortened SW VACNT	91
	DNA (guanine bases)	Guanine Oxidation	1000 nucleic acids per NEA pad		MW VACNT NEA	92
	DNA (TP53 gene mutation)	ssDNA Probe & Impedance	1.0×10−17 M		MW VACNT	45
	DNA (CEACAM5 sequence)		0.92 μM		PET modified with MW VACNT	93
Lipid	cholesterol	Polymerization of polyaniline	Not reported,	0.22 μA/mg·dL	MW VACNT	62
	simvastatin (SV)	Redox	0.01 nM	0.001 A/μM·cm2		94
glucose	glucose	Redox	100 nM	45.93 mA/mM·cm2	MW VACNT	95
	glucose		1.1 μM	620 μA/mM·cm2		96
	glucose		7.035 μM	65.816 μA/mM·cm2		97
other	glutamate	Redox	10 nM	2.2 A/mM·cm2 (for 0.01–20 μM) 0.1 A/mM-cm2 (for 20–300 μM)	VACNT NEA	69
	dopamine		0.19 μM	Not reported.	SW VACNTs glossy carbon electrode array	47
	hydrogen peroxide		0.8 μM	1.08×106 μA/M·cm2	MnO2-modified MW VACNT	98
	C-terminal telopeptide (cTx)	Antibody & Enzymatic	0.05 ng/mL	302.83 ΔZmod/ng/mL	MW VACNT modified with Au nanoparticles	99

Figure 2.

Illustrations of different sensing mechanisms: (A) VACNT sensor based on the hydrolysis of starch releasing gold nanoparticles detectable by DPSV (left) and its response to CEA (right). Reproduced with permission from ref 48. Copyright 2020 Royal Society of Chemistry. (B) Impedance spectroscopy of biotin using interdigitated VACNT electrodes. Reproduced with permission from ref 42. Copyright American Chemical Society. (C) VACNT modified FET for thrombin sensing. Reproduced with permission from ref 87. Copyright 2011 Elsevier. (D) Sensing of cancerous cells based on cell traction forces (CTF) causing measurable deflection. Here, the schematic illustrates the deflection angle of a CNT beam by the cell. Reproduced with permission from ref 50. Copyright 2013 Oxford University Press. (E) Model and SEM image of VACNT piercing cancerous cell for high resolution impedance sensing. Reproduced with permission from ref 58. Copyright 2011 Royal Society of Chemistry. (F) Nanoelectrode array (NEA) and illustration of nucleic acid binding. Reproduced with permission from ref 92. Copyright 2004 Oxford University Press.

Other nonenzymatic based VACNT protein sensors have also been well researched. As opposed to using enzymes for signal generation, these sensors utilize the innate electrical properties of CNT. Here, changes of impedance, as measured by cyclic voltammetry or electrochemical impedance sensing (EIS), indicate the target molecule capture by the covalently bonded receptor, such as an antibody or aptamer. These platforms have been used to detect a wide variety of proteins such as T-PSA,⁸⁴ CIP2A,⁸⁵ CEA,⁸⁶ and biotin⁴² (Table 1). These sensors all cited that the use of vertical alignment, rather than a random orientation, provided a myriad of advantages including: higher surface area for better electrical performance, improved functionalization because of the higher number of terminal carboxylate groups, more uniform surface arrangement for greater capture molecule immobilization, and improved electron transfer. As illustrated by this, the use of VACNT as an electrode plays a critical role in electrochemical sensors. In fact, in a recent study by Brownlee et al., electrode geometry was shown to have significant impact on sensitivity and sensing range.⁴² Compared to a serpentine design, an interdigitated design (Figure 2B) offered 1.6 times the sensitivity when detecting biotin. In another work, Croce et al. developed a novel FET sensor, which detected low levels of thrombin. Here, the VACNT acted as the gate of the MOSFET and as positively charged thrombin were captured, the gate voltage increased resulting in drain current changes (Table 1 and Figure 2C).⁸⁷ Compared to traditional electrochemical sensors, both of these sensors also improved the limit of detection by accommodating higher target capture efficiency because of the large effective surface area to volume ratio.

While these protein sensors offer either comparable or improved detection limits and sensitivities to conventional means, their importance arises from their on-chip capabilities. Unlike ELISA, these platforms can be integrated for lab-on-a-chip and even point-of-care diagnostics. Thus, if commercialized, VACNT-based protein sensors may see success in the clinical world as rapid and inexpensive devices to detect a variety of diseases.

2.1.2. Biosensors for Cancer Cells.

Early detection of cancer is paramount to patient survival. As cancer cells progress, so does their cell membrane’s electrical behavior,¹⁰⁰ surface markers,^101,102 and even structure.¹⁰³ Electrical cell–substrate impedance sensing (ECIS) can detect these by monitoring the impedance of a cell during proliferation, attachment, and movement.¹⁰⁴ However, ECIS is greatly limited by hour-long response times and needs additional adhesive layers to improve cell attachment to the electrodes. Nanobiotechnology has overcome these limitations while providing other benefits. Abdolahad et al. presented a VACNT ECIS sensor for colon cancer diagnosis, where the VACNT acted as both the adhesive and conductive layer (Table 1).⁸⁸ Metastatic cancer cells, which are easily trapped on the VACNT because of both their deformable cytoskeletal structures and the innate adhesion to VACNT, had ECIS sensitivity of 1.7 × 10⁻³ Ω cm² and limit of detection of 4000 cells/cm². More importantly, cells were detected within a few seconds, which is a great improvement over traditional ECIS. Beyond ECIS measurements, Abdolahad et al. also demonstrated that cells positioned atop VACNT result in deflection (Figure 2D).⁵⁰ The group utilized large deflection theory to calculate cell traction forces (CTF) and found that metastatic single renal carcinoma cells apply greater CTF than healthy cells and result in greater VACNT deflection. As seen in Figure 2D, the measurements were obtained by examining scanning electron microscopy (SEM) images, which limits practical use. Nevertheless, these mechanisms demonstrate the unique advantages of VACNT and how it can be used to detect early stage cancer from cell structure.

VACNTs are also able to provide high single-cell resolution sensing by penetrating the cellular membrane and measuring a cell’s impedance in vivo. This was demonstrated by Abdolahad et al., who differentiated between two metastatic stages of colon and breast cancer cells via EIS using cell-pierced VACNT electrodes depicted in Figure 2E.⁵⁸ It was observed that cells at higher metastatic stages have significantly lower impedance compared to cells in lower stages. Furthermore, Zangeneh et al. improved this mechanism by functionalizing VACNT with folic acid so that the folate receptors of lung cancerous cells enhance cell entrapment on the CNT, leading to better response.⁸⁹ The group achieved sensitivity as low as 4 × 10⁻³ Ω cm² (Table 1).

Lastly, VACNTs also exhibit high electrocatalytic behavior toward numerous biomolecules and biomarkers making them useful for developing electrochemical sensors.^45,46,64,105 VACNTs can act as a molecular nanowire enabling direct and fast transfer of electrons between the attached biomolecules and the bulk electrode.¹⁰⁶ This is favourable when measuring P-glycoprotein (P-gp) overexpression, which is responsible for the drug molecules efflux out of the cell. Targeting this molecule is important because it is an indicator for multi drug resistant (MDR), which is a major cause of chemotherapy failure.¹⁰⁷ Gulati et al. presented a flexible electrochemical immunosensor using anti-P-gp-functionalized VACNT on a PET substrate.⁹⁰ Because of VACNT’s large effective surface area, thus more binding sites and higher electrocatalytic behavior, the biosensor achieved a limit of detection as low as 10 cells/mL (Table 1).

2.1.3. Biosensors for Nucleic Acid.

Nucleic acids make up the human genetic code and are critical in the development, function, and growth of cells. For this reason, genetic mutations are a leading cause of cancer and, thus, are relevant for early cancer diagnostics.^108,109 Nucleic acid detection via fluorescent molecular beacons was introduced by Tyagi and Kramer in 1996,¹¹⁰ which inspired traditional DNA sequencing methods, such as Northern blotting,¹¹¹ microarray, and polymerase chain reaction (PCR).¹⁰⁹ However, these methods are both labor-intensive and require costly equipment. Thus, there have been significant efforts to develop highly sensitive on-chip technologies, some of which incorporate CNTs. Recent literature reports that VACNTs, in particular, can act as molecular wires and enhance electrical communication between the electrode and a redox enzyme.^105,106 This direct electron transfer between the enzyme and electrode can eliminate the need for redox mediators, making label-free nucleic acid biosensors simpler.

Wallen et al. reported a novel label-free DNA biosensor, incorporating acid-shortened single-walled VACNT on a gold substrate.⁹¹ In this biosensor, SW-VACNTs are modified with an amine-terminated, single-stranded probe-DNA. The following cDNA sequence hybridization is then monitored using electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3−/4− as the redox probe. The charge-transfer resistance decreased with time when the probe-DNA was attached to SW-VACNTs but increased when directly attached to gold surfaces. This decrease in charge-transfer resistance during the progress of hybridization indicates mediated electron transfer by the SW-VACNTs between the electrode and the redox probe in the solution. In another work, Koehne et al. presented a nanoelectrode array (NEA) made by mutli-walled VACNTs (MW-VACNTs) for rapid molecular diagnosis applications (Figure 2F).⁹² MW-VACNTs are functionalized with DNA probes of desired sequences. Guanine groups in the target DNA serve as electrochemical signal moieties, providing a small current through AC voltammetry. Since the current is very small, because of the limited number of guanine bases in the target molecules, Ru(bpy)²⁺ is employed as a mediator to amplify the signal. The detection limit of the sensor is similar to laser scanners in conventional fluorescence-based DNA microarray techiniques, thus demonstrating clinical potential (Table 1).

In the field of cancer diagnosis, Fayazfar et al. successfully detected TP53 gene mutations with a limit of detection of 1 × 10⁻¹⁷ M using a MW-VACNT forest functionalized with ssDNA probes.⁴⁵ More importantly, the group compares this sensor directly with a randomly oriented-VACNT and concludes that alignment is the key to success of the biosensor as attributed to the better electrocatalytic activity, higher surface area, and improved electron transfer. In another report, Gulati et al. detected CEACAM5, a colon cancer tumor biomarker, using ssDNA modified VACNTs on a PET substrate.⁹³ EIS results show a stepwise increase in resistance as the DNA concentration increases and cyclic voltammetry shows a decrease in the peak current when the concentration of target DNA is increased. This phenomenon is due to strong intercalation of the redox mediator methylene blue (MB) with the DNA as reducible groups of MB cause steric hindrance. The limit of detection of this device was 0.92 μM (Table 1). Perhaps the most important property of VACNT here is the ease of oxygen plasma functionalization due to the exposed tips. This allows ssDNA probes to be easily attached; without them, the sensor would lose many probing sites and, consequently, sensitivity.

2.1.4. Biosensor for Lipids.

Phospholipids, amphiphilic molecules consisting of a fatty acid tail (hydrophobic), a phosphate group (hydrophilic), and a glycerol backbone, are crucial components of the cell membrane.¹¹² Measuring lipid levels, particularly cholesterol, in human blood is clinically significant as high levels increase risk for heart disease. Traditionally, cholesterol is electrochemically measured by spectrometry via the production of a colored substance upon interaction with cholesterol molecules.^113,114 However, these methods have several disadvantages, particularly the instability or unreliability of the colored reagent and its production.

More recent research has implemented VACNTs as electrochemical biosensors since they have superb electron transfer required for monitoring the redox reaction. Specifically, Wisitsoraat et al. developed a cholesterol VACNT electrochemical biosensor that immobilizes then detects cholesterol enzymes as they polymerize a polyaniline additive. They report a sensitivity of 0.22 μA mg⁻¹ dL⁻¹ and a detection range that is suitable for sensing cholesterol concentration levels in human blood (Table 1).⁶² Also, Fayazfar et al. reported on a dihexadecyl hydrogen phosphate-modified MWCNT for the electrochemical oxidation and detection of simvastatin, an inactive lactone that can treat high cholesterol.⁹⁴ In this approach, simvastatin was detected at pharmaceutically relevant levels with a limit of detection 1000-fold lower than similar randomly oriented CNT sensors. This improved performance was owed to the larger surface area of VACNTs facilitating catalytic activity (Table 1). Furthermore, because its fabrication is compatible with conventional microfabrication techniques, this sensor can be integrated on lab-on-a-chip systems while being a rapid, reusable method to detect cholesterol, unlike its traditional colorimetric counterpart.

2.1.5. Biosensors for Glucose.

Glucose, a monosaccharide carbohydrate, serves as fuel for the body. Typically, the concentration of glucose in blood is between 80 to 120 mg/dL.¹¹⁵ However, those with diabetes are unable to metabolize glucose, resulting in glucose levels higher than the healthy range.¹¹⁶ Conventionally, blood glucose concentration is measured using enzymatic reactions with glucose oxidase. Specifically, glucose oxidase test strips react with glucose in the blood sample and produce a color change measurable by a reflectance meter.¹¹⁵ Although this technique is convenient, it has several limitations: lack of continuous monitoring, low sensitivity, and high failure rate due to user error or poor maintenance. Therefore, more novel approaches, both nonenzymatic and enzymatic, have employed VACNT-based sensors for glucose sensing.

Nonenzymatic approaches utilize a nonmodified VACNT array that, at the surface, measure oxidation of glucose. In 2018, Brownlee et al. developed VACNT in a microchannel array for flow-through electrochemical glucose sensing via the chemical reaction of glucose and methyl viologen.⁹⁵ The resultant reduction of methyl viologen followed by direct oxidation with VACNT produced a measurable current density correlating to glucose concentration. This platform offered both a low limit of detection (100 nM) and high sensitivity (45.93 mA mM⁻¹ cm⁻²) (Table 1). This nonenzymatic application is extremely suitable for VACNTs as this particular nanomaterial promotes fast electron transfer kinetics and demonstrates specificity toward glucose instead of other interfering electroactive species.

Enzymatic glucose sensors have traditionally been employed since the binding enzyme provides excellent specificity. Nevertheless, they have significant shortcomings in sensitivity and stability. VACNTs have been demonstrated as an attractive enhancement for glucose sensors as they transfer charge efficiently, have a large active surface area, and are easily functionalized with immobilization materials for sensing. In fact, Azimi et al. used a VACNT array as a sensing electrode with surface modification of electrodeposited polyaniline with subsequent covalent attachment of glucose oxidase.⁹⁶ This point-of-care glucose sensor detected glucose levels as low as 1.1 μM in human blood plasma (Table 1) and has high sensitivity of 620 μA mM⁻¹ cm⁻². Similarly, Gokoglan et al. reported on a flexible VACNT-based glucose sensor modified with poly(9,9-di(2-ethylhexyl)-fluorenyl-2,7-diyl)–end-capped with 2,5-diphenyl-1,2,4-oxadiazole as an immobilization matrix for glucose oxidase.⁹⁷ Compared to Azimi et al., the sensor had a lower limit of detection (0.735 μM) but at the cost of sensitivity (Table 1). Overall, VACNT-based glucose sensors, both nonenzymatic and enzymatic, enhance glucose sensing performance for sensitivity, specificity, and stability in comparison to traditional randomly oriented counterparts.

2.1.6. Miscellaneous Sensors (Neurotransmitters, Hydrogen Peroxide, and c-Terminal Telopeptide).

Electrochemical VACNT sensors have also been used in a variety of unique applications, including detecting analytes ranging from neurotransmitters,^69,117 to hydrogen peroxide⁹⁸ (Table 1). Glutamate, an amino acid that acts as an excitatory neurotransmitter in the central nervous system, was targeted via a VACNT nanoelectrode array by Gholizadeh et al.⁶⁹ Here, glutamate dehydrogenase was covalently bonded to the tips of VACNT and provided a low detection limit of 10 nM. Another neurotransmitter sensor for selective detection of dopamine and uric acid was developed by Yang et al.¹¹⁷ Using a unique dual oxidation electrode design comprised of single-walled VACNT arrays, the sensor was so specific that it was able to separate the oxidation potentials of dopamine and uric acid while having a limit of detection of 0.19 μM and 0.82 μM respectively. Alternatively, Xu et al. developed an amperometric sensor of VACNT modified with manganese oxide, which served as a highly efficient catalyst for hydrogen peroxide reduction.⁹⁸ This electrochemical reaction is measurable with low limit of detection (0.8 μM) and high resistance to interfering molecules. Another unique electrochemical sensor was developed to detect c-terminal telopeptide, a key biomarker in bone resorption.⁹⁹ This immunosensor uses VACNT electrodes coated with gold nanoparticles for electrochemical sensing with limit of detection 0.05 ng/mL. Compared to ELISA, this method can determine the bone turnover marker quickly and the vertically alignment of the VACNT allows controlled exposure, which controls current density thus enhancing reproducibility and precision.

2.2. Bioparticle Separation and Filtration.

With the recent advancements in microfabrication techniques, the integration of VACNTs in microfluidics, specifically for on-chip separation and filtration of biological entities, has garnered significant attention. While many groups have previously presented on carbon nanofiber filters¹¹⁸ and VACNT gas platforms,¹¹⁹ Mogensen et al. was the first to report a VACNT integrated microfluidic device for particle separation.¹²⁰ Here, using electroosmotic flow (EOF), the chip successfully separated fluorescein and 5-carboxyfluorscein. VACNT excels here since it can be biased for EOF and, more importantly, its intertubular distance allow unperturbed flow through the separation channel. This porosity will prove to be the foundation of other VACNT microfluidic platforms, namely for filtration.

Following this first report, Fachin et al. and Chen et al. published several findings on integrated VACNT in various microfluidic devices for isolation and manipulation of biological particles.^121–124 Specifically, Fachin et al. compared the flow patterns of a nonporous PDMS pillar to a highly porous VACNT pillar.¹²¹ The results (Figure 3A) show that 20 nm quantum dots rarely become trapped by the nonporous PDMS due to boundary layer effects. Meanwhile, the nanoporous VACNT improve performance by 7-fold due to the flow penetrating the structure and the quantum dots becoming lodged within the individual VACNT. Fachin et al. followed this by demonstrating that this arrangement of nanoporous VACNT could isolate particles of varying sizes, specifically 15 μm, 2 μm and 40 nm.¹²² Similarly, Chen et al. evaluated particle surface interactions of VACNT with high surface area, compared to solid surface interactions.¹²³ A 5.5-fold increase was observed by using the nanoporous VACNT. Following this report, Chen et al. studied specific enhancement of particle filtration using the ultrahigh porosity (99%) and high permeability of VACNT structures in microfluidic channels.¹²⁴ In this report, VACNT provided a 6-fold and 4-fold filtration increase for bacteria and cancer cell lines, respectively. These reports demonstrated the feasibility of such platforms and offer a direct comparison of how the porosity of VACNT is extremely advantageous compared to nonporous structures.

Figure 3.

Illustration of porous VACNT and its applications: (A) Simulation of quantum dots’ flow through nonporous structure (left) and porous CNT structure (right). The green streamline is a nonporous flow profile; the yellow is a porous flow profile through a boundary (white circle). Reproduced with permission from ref 121. Copyright 2010 IEEE. (B) Size tunable VACNT virus enrichment device illustrating the capture of large virus particles by the porous VACNT. The right panel is a typical field sample gathered via swab. The sample flows through the VACNT device (middle panel) and the virus is captured by the VACNT filter, while miscellaneous contaminants are flowed out (right panel). Reproduced with permission from ref 125. Copyright 2015 American Association for the Advancement of Science.

In more recent years, integrated VACNT research has shifted toward more novel biomarkers, such as extracellular vesicles, and more clinically relevant applications, such as viruses. Yeh et al. had tremendous contribution in this field by first reporting on a VACNT size-tunable enrichment microdevice for ultrasensitive virus detection (Figure 3B).¹²⁵ Specifically, VACNTs were engineered with size-tunability: their intertubular distances were precisely controlled between 17 and 325 nm. This tunability allowed the highly porous VACNT forest to be sized-matched to different viruses of unique size domains. The technology improved detection limits and isolation rates by 100-fold compared to traditional techniques, marking a major breakthrough in integrated VACNT microfluidics.

Since this first report, Yeh et al. published several applications including a microfluidic device of spiral-shaped VACNT forest for blood plasma extraction.¹²⁶ The device recovered 80.1% albumin from extracted plasma. In 2020, Yeh et al. had two reports on rapid, label-free, size-based isolation of both virus¹²⁷ and extracellular vesicles from clinical samples.¹²⁸ For virus capture and identification, Yeh et al. used integrated VACNT in a microfluidic platform with differential filtration porosity¹²⁷ Combined with surface-enhanced Raman spectroscopy, real-time virus capture and detection was performed with 70-fold enrichment enhancement and 90% virus specificity. For extracellular vesicle isolation, a similar VACNT microfluidic device was used to isolate extracellular vesicles derived from neurons and cells.¹²⁸ Furthermore, extracellular vesicle capture was more efficient for smaller particles than larger particles. Most recently, Jadhav et al. proposed an integrated VACNT microfluidic platform coupled with surface-enhanced Raman spectroscopy for diagnosis of COVID-19.¹²⁹ Specifically, with Raman spectroscopy of Au/Ag-VACNTs, SARS-CoV-2 could accurately be identified with enhanced sensitivity. While not yet integrated, it could act as a long-term universal virus detection system, which has large implications.

Overall, integrated VACNT in microfluidic devices, systems, and platforms has been demonstrated to enhance traditional performance. Specifically, using a microfluidic approach for separation and filtration allows for higher throughput, rapid performance and, typically, easier use, due to compact, miniaturized, automation, and user-friendly design. Furthermore, as advancements continue to be made in carbon nanomaterial synthesis techniques, as well as micro- and nanofabrication, better device performance and expanded applications will appear.

2.3. Cell Stimulation and Recording.

The nervous system is a highly complex network of cells that communicate via electrical signals.¹³⁰ Recording these signals can provide a better understanding of neurological diseases and pave the way for treatment and therapy.¹³¹ Neural electrodes are traditionally fabricated using noble metals, such as gold and platinum, since they are chemically inert.¹³² Nevertheless, their low charge injection limit, delamination, and particle deposition under high current density make them imperfect implantable electrodes. While improved sensitivity and charge injection can be overcome by enlarging electrode geometry, invasiveness and tissue damage will also increase.¹³³ Thus, CNT, with its great electrical conductivity, large surface area, and high aspect ratio has aroused tremendous interest. VACNTs offer even greater advantage with their ability to penetrate inner parts of a cell, without membrane damage, greatly improving signal resolution.^58,134 Furthermore, as mentioned, VACNTs offer better electron transfer than randomly oriented CNTs. Also, CNTs are mechanically rigid enough to be inserted deep into tissue, yet have slight flexibility thus can move with natural tissue vibrations without causing extensive cell damage.⁶⁵ This makes the nanomaterial exemplary for use in microelectrode arrays (MEA) for cell electrical-stimulation and recording.

The first VACNT MEA for neural interface was developed in 2006 by Wang et al.¹³⁵ VACNTs were synthesized on a poly-silicon/quartz substrate, then partially encapsulated by silicon dioxide, silicon nitride, and silicon dioxide, respectively. The electrode successfully stimulated embryonic rat hippocampal neurons with high charge injection limit of 1–1.6 mC/cm². More importantly, the protruding geometry of the VACNT was key in penetrating the layered tissue. Furthermore, Wang et al. demonstrated that implanting in this way, as opposed to growing conformally on the electrode, provided better proximity between the electrode and the cells.¹³⁵ This makes neural stimulation much safer as it requires less current and thus less heat is generated in the surrounding tissue. In another work, Keefer et al. coated conventional tungsten and stainless-steel wire electrodes with VACNT to evaluate their effect on electrical recording and stimulation on the visual cortex area of monkeys (Figure 4A).¹³⁶ It was reported that the VACNT improved the charge transfer by 40-fold and lowered impedance by 23-fold. Similar to Wang et al., the vertical dimension of VACNT in the work by Keefer et al. permitted the electrode tip to penetrate multiple layers of cell debris.^135,136 The rough surface of the electrode also produced excellent cell-electrode coupling, which improved the signal-to-noise ratio.

Figure 4.

(A) Conventional tungsten electrode modified with VACNT and corresponding local field potential traces (top) and power spectral density (bottom) demonstrating the superiority of VACNT modification. Reproduced with permission from ref 136. Copyright 2008 Springer Nature. (B) Illustration of flexible actuator with VACNT electrode for cultivation and stimulation of cardiac tissue layer; displacement plot of VACNT over time under electrical stimulation. Reproduced with permission from ref 65. Copyright John Wiley and Sons.

Tian et al. employed VACNTs sandwiched between two flexible parylene C layers to develop a novel cuff electrode for peripheral nerve stimulation (PNS).¹³⁷ The flexible cuff electrode was tested to stimulate rat sciatic nerves and showed better performance compared to Pt electrodes. This was largely due to the penetrating VACNT-electrodes increasing the selectivity by filling in gaps that traditionally exist between the cuff electrode and nerve. Rafizade-Tafti and Abdolahad utilized VACNT as a nanosize field enhancer to study the bioelectrical changes in cells upon electromagnetic stimulation.^138,139 In their work, lung cancer cells were cultured on VACNTs whose tips penetrated their cell membrane. Upon electromagnetic wave irradiation, cells experienced electrochemical variations that lowered their impedance. Furthermore, they combined this impedance-based biosensor and transducer for EM stimuli in one system to manage the transmembrane charge and achieve controllable apoptosis.¹³⁹ In another example, Shin et al. designed a flexible tissue actuator with VACNTs MEAs integrated in gelatin methacrylate (GelMA) bonded on a flexible hydrogel layer (Figure 4B).⁶⁵ Cardiomyocytes were then cultured on the CNT-GelMA hybrid hydrogel surface to induce maturation of cardiac muscle tissues. The beating frequency of this 3D biohybrid actuator could be controlled via the electrical pathway of the VACNT. The group reports that such electrodes would have not been possible using only randomly oriented CNT dispersed in a gel as VACNT allows close contact to the cells, enabling localized stimulation at low voltages. Interestingly, because of the vertical alignment of the CNT, significantly different excitation thresholds could be observed for electric field in the parallel and perpendicular directions relative to VACNTs. This anisotropic conductivity better mimics the conductivity presented in the fiber structure of cardiac muscle tissue. The works presented here demonstrate the advantages of using VACNT for cell stimulation and recording. Many report that such MEA platforms are more suitable than traditional noble metal electrodes since they have larger charge injection, can pierce cells for better resolution and are less damaging. Thus, VACNTs have proven to be an excellent nanomaterial for the ongoing investigation in neural therapy.

2.4. Drug Delivery via Microneedles.

Most transdermal drug delivery methods, such as vaccination or biotherapeutics, are facilitated via needles. This method offers a low cost, rapid, and direct way of injecting target substances into the body. However, the injection is painful, must be administrated by trained personnel, and is nondisposable.^140,141 Microneedles resolve many of these limitations; they are less painful, less likely to cause needle-prick injuries, and better to protect against microbial infection since the remanent puncture in the skin is microscopic.^140,141 In particular, hollow microneedles, which operate under the same pressure-driven mechanism as traditional needles, have become the target of VACNT-based drug delivery systems. Specifically, VACNT’s high aspect ratio and hollow center make them great candidates for both loading and delivering drugs. In fact, this hollow center makes them excellent drug delivery systems beyond just microneedles. For example, drugs can be loaded in the cavity of the CNT and capped with nanoparticles. Then the drug-loaded CNT can be delivered to the target site by penetrating the cell membrane, where the ends can be cleaved, thereby releasing the drugs.^25,27 The cleaving mechanism is often a stimulus-based event, such as a pH change. These systems, however, are not vertically aligned and have already been heavily reviewed so they will not be discussed in detail.²⁷ Here, unique applications of VACNT in drug delivery will be discussed. These methods demonstrate how the unique mechanical and structural properties of VACNT are advantageous over their randomly oriented counterpart for transdermal delivery of drugs and vaccines.

The integration of VACNT in microneedles began in 2014 when Wang et al. used the nanomaterial as a control switch for drug delivery.¹⁴² VACNT filters were fabricated and enclosed within an SU8 microneedle (Figure 5A). After synthesizing VACNT via CVD, the intertubular space was filled with parylene-C for additional support. Then, the entire structure was removed from the substrate and both ends of the VACNT were opened via a plasma etch. This allowed for drug loading as described earlier. Finally, the SU8 hollow microneedle was fabricated by a double drawing lithography process and covered in a gold film and attached to a drug containing vessel. Here, the role of VACNT is 2-fold: it mechanically filters all particles larger than the inner diameter of the VACNT and serves as a gate for drug injection. For example, the group loaded insulin into the vessel and then applied pressure which forced the insulin through the hollow center of the VACNT and out the microneedle tip. Furthermore, since insulin is a charged molecule, they also demonstrated that, by utilizing the gold coating of the microneedle, an electric field could be created between the drug solution and the target delivery site. Because VACNTs exhibit excellent latitudinal electrical conductivity, a positive bias promotes the travel of insulin through the hollow centers and into the target site, while a negative bias decreases delivery. Thus, the system can act as both a pressure and electric field valve for insulin delivery.¹⁴²

Figure 5.

Illustration of various VACNT integrated drug delivery systems: (A) Array of voltage-gated microneedles integrated with a VACNT filter. Reproduced with permission from ref 142. Copyright 2014 IEEE. (B) Illustration of VACNT microneedle with polyimide composite with corresponding on-skin platform for transdermal injection of drugs. Reproduced with permission from ref 43. Copyright 2014 Springer Nature.

In another paper, Lyon et al. fabricated a microneedle from VACNT.⁴³ Traditionally, hollow microneedles have challenging fabrication protocols. These often involve many sacrificial substrates, wet chemical etching, expensive equipment, multiple lithography steps, and lengthy processes. However, this innovative fabrication protocol involves a single lithography step to pattern the microneedle array, CVD to synthesize the VACNT, and a simple spin coating and curing of polyimide to provide structural stability. Here, the polyimide is critical as it makes the CNT microneedle nonporous to hold drugs and allows for separation of the VACNT from the single sacrificial substrate and adhesion onto a microfluidic platform (Figure 5B). The platform successfully injected methylene blue dye into swine skin and showed clear potential as a cheap, disposable, and easy to fabricate microneedle platform for transdermal drug delivery, such as insulin.⁴³

2.5. Scaffolding: Cell Adhesion and Migration.

Nanotopography greatly influences the adhesion of cells to a particular substrate. This idea has been supported since the 1990s with research of textured, high aspect ratio silicon grass used to attract cortical astrocytes.¹⁴³ This effect is most notably due to the resemblance of extracellular matrix, mimicking the in vivo environment of cells, and promoting their growth. Other topographical cues, such as directional physical signals transmitted from adhesion to actin filaments, also aid in its effectiveness.¹⁴⁴ As described earlier, CNTs have a high aspect ratio (>1:1000), making them ideal candidates for such scaffolding. Moreover, MWCNT diameter can be similar to the dimensions of ECM proteins making it even more promising.³⁶ Compared to conventional synthetic polymer scaffolds, CNT networks do not present concerns with toxicity and have high mechanical strength to withstand cell force during growth.¹⁴⁵ Also, CNTs are conductive and easily patternable, making them suitable for selective cell growth and stimulation. This is particularly advantageous compared to natural scaffold materials such as collagen. These characteristics make it a superior candidate in certain tissue engineering applications.

It was not until the 2000s, when the production of CNT became widely accessible, that the nanomaterial saw use in cell scaffolding.⁶⁰ At this time, however, the synthesis technology was in its infancy and pristine VACNT without amorphous carbon was unachievable in situ. Thus, the uses of CNT as scaffolding for neurons was achieved using randomly dispersed functionalized-CNT islands.⁶⁰ Shortly after, in 2005, Zhang et al. utilized VACNT for neurite growth and the benefits of VACNT over randomly oriented CNT for neuron cultivation became truly apparent. Specifically, they reported that neuron cells preferentially attach to the sidewalls of VACNT islands.¹⁴⁶ As stated earlier, VACNTs have much higher surface areas, better charge injection capability, and pattern-ability, thus making them more suitable as electrodes, which can self-assemble neural networks. This same conclusion was presented by Nick et al. and supports that VACNTs can be used to culture neuron cells and simultaneously stimulate and record their action potentials.¹⁴⁷

Beyond neuron cultivation, there have been several studies conducted on the viability of VACNT for tissue engineering (Table 2). One early demonstration of this was in 2004 with the cultivation of fibroblast cells on structurally modified VACNT.¹⁴⁵ Here, the VACNT was acid treated to create a cross-linked 3D hydrophilic sieve network which segregated cells and favored growth. This work was expanded upon by Lobo et al., who primarily utilized VACNT for fibroblast and osteoblast cell culturing. In 2008, they successfully adhered fibroblast L929 mouse cells onto the tips of nonmodified VACNT.^70,148 Here, the key was the in situ growth of VACNT via CVD, which removed amorphous carbon that may have lowered biocompatibility. At the time, this study was valuable as the cytotoxicity of nonfunctionalized CNT was not well understood. Recently, a few review papers have discussed in detail the toxicological profile of CNT, which generally depends on purity, length, functionalization, and molecular conjugation.²⁹ It was determined that the pristine condition and long length of in situ grown VACNT provide suitable conditions for cell adhesion and growth.^70,148 However, as VACNT is superhydrophobic, it naturally has aversions to cell adhesion and migration, thus limiting the applications of VACNT in cell-related work.

Table 2.

Summary of VACNT Scaffolds for Tissue Engineering

year	cell	scaffold	ref
2000	neuron	4-hydroxynonenal (4-HNE)-CNT	60
2004	mouse fibroblast L929	3D acid treated VA-MWCNT	145
2005	neurites	poly-l-lysine (PLL) VA-MWCNT	146
2008	mouse fibroblast L929	VA-MWCNT	148
2011	mouse fibroblast L929	VA-MWCNT-oxygen plasma (O2)	36
2013	human osteoblast	nanohydroxyapatite (nHAp)/VA-MWCNT-O2	150
2013	chondrocytes	VACNT-O2	37
2013	mesenchymal stem cells (MSCs)	collagen/VA-MWCNT	154
2013	mouse precursor muscle cells	VA-MWCNT	155
2014	Tritrichomonas foetus (T. foetus)	VA-MWCNT-O2	153
2014	neurons	VACNT	147
2015	human osteoblast	poly(d,l-lactic acid)/VA-MWCNT-O:nHAp	158
2016	retinal precursor (R28)	VA-MWCNT-O2	156
2017	human osteoblast	electrospun poly lactic acid/VA-MWCNT-O:nHAp	151
2019	chondrocytes	porous poly(d,l-lactic acid) /VA-MWCNT-O:nHAp	152
2020	retinal neurite	VACNT atop aluminum electrode	157

In 2010, the group addressed the hydrophobicity issue by treating the CNT with oxygen plasma, transforming the material from superhydrophobic to superhydrophilic.¹⁴⁹ As stated earlier, VACNTs are much easier to functionalize than randomly oriented CNTs due to the larger number of defects at the tips.^{35–37,61,66,67} After demonstrating the biocompatibility of the oxygen plasma treated VACNT scaffolds, it was shown that the oxygen plasma functionalization accelerated the adhesion and increased the proliferation of fibroblast L-929 cells and chondrocytes, as visualized in Figure 6.^35–37 The superhydrophilic pristine-VACNT scaffold fostered a wide variety of tissue engineering applications. Later, the scaffold was modified with hydroxyapatite (nHAp), a phosphate biomaterial similar to natural bone, to promote the growth of chondrocytes.¹⁵⁰ Here, the superhydrophilic functionalization of CNT is mandatory for the deposition of nHAp. This work was furthered by Rodrigues et al. and Stocco et al., who transferred the scaffold to electrospun poly(lactic acid) and polycaprolactone fibers, respectively, for osteochondral and meniscus tissue engineering.^151,152 Other therapeutic applications also came about, including the use of oxygen plasma treated VACNT scaffolds for photodynamic therapy to cultivate and then eradicate Tritrichomona foetus parasite.¹⁵³ This technique shows potential for the usage of VACNT in tumor treatment.

Figure 6.

SEM images of cell proliferation on VACNT. Reproduced with permission from ref 37. Copyright 2013 Elsevier.

During this time, other groups also reported their work on VACNT scaffolds. Bitirim et al. investigated mesenchymal stem cells (MSC) growth on noncoated and collagen coated VACNT.¹⁵⁴ The scaffold was patterned to create nest-like structures that were suitable for the MSC proliferation. The work demonstrated potential for transplantation therapy applications or regenerative therapy for central nervous system injuries. Furthermore, Holt et al. demonstrated that VACNT can be used as a biocompatible substrate for directional alignment of mouse muscle cells where, similar to neuron cells, the cells grow on the edges of the nanotubes.¹⁵⁵ Lastly, there are reports of culturing retinal cells on VACNT as a dual scaffold and implantable MEA.^156,157 It was reported that the mechanical strength of VACNT was critical for retinal neurite outgrowth and the high surface area made for a high-quality electrode. All VACNT scaffolds are summarized in Table 2.

2.6. Bactericide and Biofilm.

2.6.1. Bactericide.

In 2007, Kang et al. investigated the interaction of randomly oriented SWCNTs and microbes. In their work, they suggested that the narrow diameter (1 nm) of the nanotube enabled it to penetrate the cellular membrane resulting in irrecoverable damage to bacteria.^55,56 This conjecture was similar to one made by Kostarelos et al., who proposed the idea of drug encapsulated CNT delivery systems discussed earlier.²⁵ However, this hypothesis has primarily been supported for CNTs dispersed in a liquid medium, as opposed to VACNTs, since CNTs in these mediums are mobile and are able to pierce cells at a high rate. Moreover, it has significant drawbacks compared to using a solid substrate for antimicrobial activity. Specifically, a large surface area of CNT is required for microbe agglomeration, but this can be difficult to achieve since CNTs often aggregate with themselves.⁴⁴ As an alternative, recent investigations have turned to VACNT as antibacterial surfaces since they are more easily synthesized and functionalized than high concentration suspensions. In fact, VACNTs have distinct anisotropic benefits, easier functionalization, simpler fabrication, and even elastic properties that make them well suited for antimicrobial applications.

An early study of VACNT for bactericide was conducted by Akhavan et al. In this work, VACNTs were synthesized and silver nanoparticles were deposited on its tips.⁵⁷ Silver nanoparticles bind to and disrupt the membrane of the bacteria by generating reactive ion species, which are detrimental to bacteria. In this study, the nanoparticle-modified tips make direct contact with the bacteria via a dropcast method and utilize the antibacterial property. In another report, Linklater et al. hypothesized a new bactericidal mechanism based on cicada wings, which have nanopillar structures similar to VACNTs.⁵² They concluded that the bactericide activity of VACNTs is based on its elastic property. As bacteria rests on VACNTs, the structure begins to bend, creating elastic energy that stretches the bacteria and results in death (Figure 7). Here, short VACNTs were determined to have greater elastic energy and improved bactericide. In fact, 1 μm high VACNTs were found to have 1000× more elastic energy than 30 μm high VACNTs, according to Euler and Bernoulli’s beam theory. For the taller VACNT structures, the bactericide rate was less than 20%, which was comparable to another report that investigated biofilm buildup on a 20 μm structure.^52,159 However, the shorter 1 μm structure achieved bactericide rates as high as 99.3% due to its much larger elastic energy. Thus, bacterial toxicity is not only based on diameter (bacteria penetration) and functionalization (Ag nanoparticles), but also length (elasticity).

Figure 7.

Illustration of VACNT deflection and plot of corresponding deflection angle to relative stored elastic energy under presence of bacteria at various VACNT height. Reproduced with permission from ref 52. Copyright 2018 American Chemical Society.

2.6.2. Biofilm.

VACNTs have also been researched toward biofilm prevention. Biofilms are generally characterized as the buildup of bacteria, and despite having numerous benefits, such as degrading toxic compounds, they can be harmful to implanted devices, quickly leading to infections.¹⁶⁰ While bactericide rates are based on cell death, biofilm is largely concerned with bacteria build up as measured by volume. Malek et al. tested the formation of biofilm on two different heights of VACNTs (407 and 540 μm) and concluded that taller VACNTs were better able to prevent biofilm formation.⁵³ This is because taller VACNTs are more elastic and mobile and prevent bacteria build up better. An alternative explanation is that the VACNT nanotopography prevents the buildup of bacteria since the tips are too small for bacteria to adhere to it properly. This proposed mechanism is similar to another report, which utilized VACNT to decrease platelet adhesion on blood contact devices.¹⁶¹ In both cases, VACNTs were capable of acting as antiadhesion devices, which is counterintuitive to the cell scaffolds previously discussed. This observation demonstrates a valuable point: the therapeutic application of VACNT is largely determined by the interacting biospecies, the functionalization, and the physical structure of the CNTs.

3. CONCLUSION

3.1. Outlook.

VACNT-related research remains of great interest to many scientific communities with new discoveries and technologies continually being made. This includes exploring the numerous vague understandings regarding VACNTs, specifically with respect to their biocompatibility and toxicity. To effectively utilize VACNTs in biomedical applications, VACNTs must be clinically integrated with superb biocompatibility and minimal or no toxicity. Many studies have focused on biocompatibility of randomly oriented CNTs. However, as VACNTs are being used in tissue engineering and cell interfacing interactions, efforts in understanding and promoting VACNT biocompatibility will become necessary. Moreover, the elusiveness of biocompatibility has placed clear restrictions on VACNTs’ usage in clinical settings, especially with implantable technologies. With the aforementioned advantageous properties of VACNTs, including mechanical, electrical, and chemical, VACNTs show great future potential in applications such as for implantable neural stimulation and recording, sophisticated drug delivery and therapeutics, and in situ sensing. Future trends in understanding and improving CNT biocompatibility and toxicity will enable implantable VACNT technologies with feasible clinical integration.

Another growing future trend in VACNTs for biomedical applications is in vitro applications, particularly in diagnostics and sensing. Diagnostically, research is ongoing in cancer-related and virus-related applications. Specifically, with the recent COVID-19 pandemic, many publications have focused on highly sensitive, rapid viral detection. However, even the most recent publications still require tedious sample preparation and downstream analysis. Furthermore, the devices are single use and not universal for all viruses. In a journal published this year, a prototype for a long-term reliable SARS-CoV-2 detection chip was proposed.¹²⁹ This VACNT chip utilizes Raman Spectroscopy and machine learning to analyze signals of various known virus molecules. Thus, it could also be a universal detection platform and potentially even multiplex virus detection. Utilizing VACNT in these types of applications could be the key to overcoming numerous technical challenges in future efforts.

In sensing, a more novel application has arose over the past few years: VACNT-based wearable sensors. Specifically, a flexible pressure sensor of sandwiched VACNT carpets embedded in polydimethylsiloxane (PDMS) substrates was used to detect heart rate, muscle flexing, and walking signals.¹⁶² This sensor demonstrated stability with 10 000 cycles and capability for long-term use. A secondary robust, stretchable, wearable sensor was developed via VACNT and PDMS thin films.⁵¹ This sensor detects electromechanical performances of the human body with ultrasensitive capabilities. Additionally, research trends are focusing on how to use VACNT to detect extremely novel and, even rare, biomarkers indicative of many disease types or states.

3.2. Commercialization.

In all future biomedical trends, one focus remains constant—commercialization. CNTs have been commercialized in a variety of air and water filtration, aerospace, and transportation, consumer and industrial goods, and energy applications.¹⁶³ In fact, more than 1000 tons of CNTs are produced globally each year.¹⁶³ However, minimal efforts have been made in commercializing VACNTs for biomedical applications. A significant challenge for commercial-level production of VACNT is controlled, reliable, and high-throughput synthesis. Current technical limitations have prevented many successes, however, research efforts are being made to improve this technical limitation.¹⁶⁴ Moreover, challenges in VACNT-to-substrate transfer methods have made VACNT integration into devices limited.

In general, the patent landscape of CNTs has been a growing field in the last 20 years. By 2010, there were 1800 patents being submitted per year with that number continuing to increase.¹⁶⁵ However, according to Google Patents on December 2021, the number of patents on “VACNT,” “vertically aligned carbon nanotubes,” and “aligned carbon nanotubes” were much smaller with approximately 600 patents in the last five years. Narrowing that search to sensing or diagnostics, the number reduces to nearly 250 and 100, respectively. Thus, a patent review further suggests that commercialization may be limited, but allows for great potential. Furthermore, continual and future advancements in micro/nanofabrication capabilities, 3D nanomaterial synthesis techniques, and device engineering will make VACNT commercialization a reality.

3.3. Summary.

Since the discovery of CNTs in 1991, this nanomaterial has shown tremendous novelty in a variety of fields including basic science, engineering, and biomedical science. Specifically, as the basic understanding of CNT’s physical properties were gained, including their atomic arrangement and self-organization during synthesis, the CNT-related research rapidly advanced. With a variety of different VACNT synthesis mechanisms and techniques, including arc discharge and chemical vapor deposition, VACNT synthesis can be highly customizable and specific with fine-tuning and precise control. Additionally, VACNTs, unlike their randomly oriented CNT counterparts, significantly improve the surface-area to volume ratio.

Further research revealed the unique mechanical, electrical, and chemical properties of CNTs. Mechanically, VACNTs demonstrate extremely high tensile strength, traditional with CNTs, with the added benefit of elasticity because of their alignment. Additionally, the tips of VACNTs are electrically active contact points that can be used in numerous sensing applications. Lastly, VACNTs are extremely susceptible to chemical functionalization on both the wall surface and tips. Chemical modification of VACNTs can be critical to biocompatibility as well as sensing and delivery applications. Overall, this highly oriented, high surface area nanomaterial offers many advantages over its randomly oriented counterpart and is more favorable in diagnostic and therapeutic applications.

Here, VACNTs were presented and reviewed under a variety of different applications. Specifically, VACNT-based electrochemical sensors have been used to detect proteins, cells, nucleic acids, phospholipids, and sugars, which demonstrated higher sensitivity and specificity compared to randomly oriented CNT electrochemical sensors. Additionally, VACNTs have been integrated into microfluidic platforms for mechanical filtration, employed as electrodes for stimulation and recording of excitable cells, unique drug delivery systems, tissue engineering constructs for therapeutic purposes, and antibacterial scaffolds.

Furthermore, this Review has demonstrated that the unique and highly specific orientation of VACNTs can be beneficial, and even better than randomly oriented CNTs, in biomedical applications. In particular, spatial arrangement and customizable VACNTs have allowed for the exploitation of their mechanical, electrical, and chemical properties to achieve higher performance than traditional technologies. Additionally, as improvements in synthesis and technology development continue to be made and clinical demands continue to grow, VACNTs could serve as an excellent solution to many clinical problems. It is foreseeable that VACNTs will be further developed into wearable sensors and highly efficient point-of-care detection and therapeutic technologies with great commercialization potential.

ABBREVIATIONS:

VACNT

vertically aligned carbon nanotube

CNT

carbon nanotube

MWCNT

multiwalled carbon nanotube

SWCNT

single-walled carbon nanotube

CVD

chemical vapor deposition

PECVD

plasma enhanced chemical vapor deposition

DEP

dielectrophoresis

ELISA

enzyme-linked immunosorbent assay

CEA

carcinoembryonic antigen

DPSV

differential pulse stripping voltammetry

EIS

electrochemical impedance sensing

ECIS

electrical cell–substrate impedance sensing

CTF

cell traction forces

SEM

scanning electron microscopy

P-gp

P-glycoprotein

MDR

multi drug resistant

PCR

polymerase chain reaction

NEA

nanoelectrode array

MB

methylene blue

EOF

electroosmotic flow

MEA

microelectrode arrays

PNS

peripheral nerve stimulation

GelMA

gelatin methacrylate

MSC

mesenchymal stem cell

nHAp

hydroxyapatite

PDMS

polydimethylsiloxane