Although many advanced biosensing techniques have been purposed for cytokine profiling, there are no clinically available methods that integrate high-resolution immune cell monitoring and in situ multiplexed cytokine detection together in a biomimetic tissue microenvironment. The primary challenge arises due to the lack of suitable label-free sensing techniques and difficulty for sensor integration. In this work, we demonstrated a novel integration of a localized-surface plasmon resonance (LSPR)-based biosensor with a biomimetic microfluidic ‘adipose-tissue-on-chip’ platform for an in-situ label-free, high-throughput and multiplexed cytokine secretion analysis of obese adipose tissue. Using our established adipose-tissue-on-chip platform, we were able to monitor the adipose tissue initiation, differentiation, maturation and simulate the hallmark formation of crown-like structures (CLS) during pro-inflammatory stimulation. With integrated antibody-conjugated LSPR barcode sensor arrays, our platform enables simultaneous multiplexed measurements of pro-inflammatory (IL-6, and TNF-α) and anti-inflammatory (IL-10, and IL-4) cytokines secreted by the adipocytes and macrophages. As a result, our adipose-tissue-on-chip platform is capable of identifying stage-specific cytokine secretion profiles from a complex milieu during obesity progression, highlighting its potential as a high-throughput preclinical readout for personalized obesity treatment strategies.

Circulating tumor cells (CTCs) carried by the patient's bloodstream are known to lead to the metastatic spread of cancer. It is becoming increasingly clear that an understanding of the nanomechanical characteristics of CTCs, such as elasticity and adhesiveness, represents advancements in tracking and monitoring cancer progression and metastasis. In the present work, we describe a combined microfluidic-atomic force microscopy (AFM) platform that uses antibody-antigen capture to routinely isolate and nanomechanically characterize CTCs present in blood samples from prostate cancer patients. We introduce the reversible assembly of a microfluidic device and apply refined and robust chemistry to covalently bond antibodies onto its glass substrate with high density and the desired orientation. As a result, we show that the device can efficiently capture CTCs from patients with localized and metastatic prostate cancer through anti-EpCAM, anti-PSA, and anti-PSMA antibodies, and it is suitable for AFM measurements of captured intact CTCs. When nanomechanically characterized, CTCs originating from metastatic cancer demonstrate decreased elasticity and increased deformability compared to those originating from localized cancer. While the average adhesion of CTCs to the AFM tip surface remained the same in both the groups, there were fewer multiple adhesion events in metastatic CTCs than there were in their counterparts. The developed platform is simple, robust, and reliable and can be useful in the diagnosis and prognosis of prostate cancer as well as other forms of cancer.

In atomic force microscopy (AFM) investigations, knowledge of the cantilever tip radius R is essential for the quantitative interpretation of experimental observables. Here we propose two techniques to rapidly quantify in-situ the effective tip radius of AFM probes. The first method is based on the strong dependency of the minimum value of the free amplitude required to observe a sharp transition from attractive to repulsive force regimes on the AFM probe radius. Specifically, the sharper the tip, the smaller the value of free amplitude required to observe such a transition. The key trait of the second method is to treat the tip–sample system as a capacitor. Provided with an analytical model that takes into account the geometry of the tip–sample’s capacitance, one can quantify the effective size of the tip apex fitting the experimental capacitance versus distance curve. Flowchart-like algorithms, easily implementable on any hardware, are provided for both methods, giving a guideline to AFM practitioners. The methods’ robustness is assessed over a wide range of probes of different tip radii R (i.e. 4 < R < 50 nm) and geometries. Results obtained from both methods are compared with the nominal values given by manufacturers and verified by acquiring scanning electron microscopy images. Our observations show that while both methods are reliable and robust over the range of tip sizes tested, the critical amplitude method is more accurate for relatively sharp tips (4 nm < R < 10 nm).

In this work, for first time, circulating tumor cells (CTCs) are captured on an open biofunctionalized substrate with multiplexing capability. This is achieved by developing a new microfluidic probe (MFP) integrated with radially staggered herringbone (HB) elements for microvortex generation. The new tool, named as herringbone microfluidic probe (HB‐MFP), is a channel‐less microfluidic system with physically separated bottom capture substrate and top fluidics delivery system. The concept allows for functionalizing the capture substrate with multiple biorecognition ligands (in this work, stripes of different capture antibodies) and scanning the fluidics delivery system across the substrate in a 2D printing‐like movement. Using the HB‐MFP, CTCs are efficiently captured from prostate cancer blood samples through their specific EpCAM, PSMA, and PSA antigens in a single run, with counts ranging from as low as 6 CTCs mL‐1 (localized cancer patients) to as high as 280 CTCs mL‐1 (metastatic cancer patients). In the process, CTC clusters with sizes of as high as 40–50 cells are also successfully captured. The results indicate that multiplex profiles of CTCs could reveal certain cellular phenotypes based on PSMA and PSA expression levels. The developed HB‐MFP is simple and robust to use, allows for high throughput sample processing, and provides seamless access to captured CTCs for further downstream characterization.

A study of the validity of analytical methods for calculating the electrostatic force interaction in alternating current electrostatic force microscopy is presented. Using a simple harmonic oscillator model, two analytical frameworks aimed at relating the electrostatic force between the cantilever tip and the sample with measurable parameters (amplitude and phase of the cantilever) are derived. The validity of the frameworks is examined based on two parameters that define the oscillation amplitude of the cantilever (tip voltage and tip-sample distance). Results are compared with an analytical model of the electrostatic interaction between tip and sample (tip-sample capacitance) and the range of validity of these two frameworks is provided. Our analysis confirms that the commonly used interpretation of the amplitude and the phase as a measure for the electrostatic force and for the derivative of the electrostatic force is only valid for very small oscillation amplitudes and depends on the tip geometry. Furthermore, this study demonstrates that these two techniques suffer from sensitivity limitations at large tip-sample distances. Finally, we compare the two frameworks with an alternative technique for the quantification of the tip-sample electrostatic interaction we have recently proposed and we discuss and experimentally demonstrate its advantages in terms of reliability and sensitivity, providing an example of dielectric constant measurement of a thin insulating film.

3D Generation of Multipurpose Atomic Force Microscopy Tips In article number 2201489, Ayoub Glia, Muhammedin Deliorman, and Mohammad A. Qasaimeh report on the technology of 3D generation of multipurpose atomic force microscopy tips (3DTIPs). 3DTIPs are developed with great flexibility in material, design, integration, and function. 3DTIPs are capable of obtaining high resolution and high‐speed atomic force microscope imaging using common modes, well suited for various applications in air and liquid, and can significantly stimulate advances in various fields. 3DTIPs might serve as a milestone in advancing scanning probe microscopy from a mire common imaging tool towards an evolved platform integrable with broader research applications.

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In this work, 3D polymeric atomic force microscopy (AFM) tips, referred to as 3DTIPs, are manufactured with great flexibility in design and function using two‐photon polymerization. With the technology holding a great potential in developing next‐generation AFM tips, 3DTIPs prove effective in obtaining high‐resolution and high‐speed AFM images in air and liquid environments, using common AFM modes. In particular, it is shown that the 3DTIPs provide high‐resolution imaging due to their extremely low Hamaker constant, high speed scanning rates due to their low quality factor, and high durability due to their soft nature and minimal isotropic tip wear; the three important features for advancing AFM studies. It is also shown that refining the tip end of the 3DTIPs by focused ion beam etching and by carbon nanotube inclusion substantially extends their functionality in high‐resolution AFM imaging, reaching angstrom scales. Altogether, the multifunctional capabilities of 3DTIPs can bring next‐generation AFM tips to routine and advanced AFM applications, and expand the fields of high speed AFM imaging and biological force measurements.