Traditional electrochemiluminescent (ECL) bioanalysis suffers from the demand for excessive external coreactants and the damage of reaction intermediates. In this work, a poly(ethylenimine) (PEI)-coupled ECL emitter was proposed by covalently coupling tertiary amine-rich PEI to polymer dots (Pdots). The coupled PEI might act as a highly efficient coreactant to enhance the ECL emission of Pdots through intramolecular electron transfer, reducing the electron transfer distance between emitter and coreactant intermediates and avoiding the disadvantages of traditional ECL systems. Through modification of the PEI-Pdots with tDNA, a sequence partially complementary to cDNA that was complementary to the aptamer of target protein biomarker (aDNA), tDNA-PEI-Pdots were obtained. The biosensors were produced using Au/indium tin oxide (ITO) with an aDNA/cDNA hybrid, and an ECL imaging biosensor array was constructed for ultrasensitive detection of protein biomarkers. Using vascular endothelial growth factor 165 (VEGF165) as a protein model, the proposed ECL imaging method containing two simple incubations with target samples and then tDNA-PEI-Pdots showed a detectable range of 1 pg mL-1 to 100 ng mL-1 and a detection limit of 0.71 pg mL-1, as well as excellent performance such as low toxicity, high sensitivity, excellent selectivity, good accuracy, and acceptable fabrication reproducibility. The PEI-coupled Pdots provide a new avenue for the design of ECL emitters and the application of ECL imaging in disease biomarker detection.

胰腺癌是一种恶性程度极高的消化道肿瘤,90％以上为起源于导管上皮的胰腺导管腺癌,早期诊断和治疗困难,预后差,在发达国家恶性肿瘤致死率中居第4位,5年生存率＜5％,是预后最差的恶性肿瘤之一[1-3].由于胰腺癌早期诊断困难,大部分患者在确诊后已经失去了手术时机,只有不到20％的患者有手术切除治疗的机会[4].随着细胞分离和鉴定技术的发展,外周血中循环肿瘤细胞(circulating tumor cell,CTC)检测成为可能,为胰腺癌的早期诊断、个体化治疗方案的选择及预后评估提供了新的方法.本文主要就CTC的检测方法及其在胰腺癌诊疗上的研究进行综述.

Ultrasound (US) needle tracking with a real-time, portable imaging system is common for biopsies and therapeutic injections. However, proper US probe-needle orientation is required or tracking precision is lost because of poor US contrast and additional artifacts. In contrast, the strong photoacoustic (PA) signal from a needle is relatively independent of light orientation. Unfortunately, a bulky, slow (10s of Hz) laser is usually required for sufficient pulse energy at centimeters depth inside the body, hindering PA integration into a real-time US scanner. We employ a compact, low-cost, low pulse energy (1 mJ/pulse) laser that can operate at 10s of kHz to construct an integrated US/PA system with frame rates higher than 30 Hz. A scanning approach with a rotating galvo mirror was used by combining multiple laser shots covering a large scan region to form an integrated image. Multiple pulse-echo US focused beams were formed between laser firings. Insertion of an 18-gauge needle in a piece of chicken breast tissue, and subsequent injection of an absorptive agent through the needle, was imaged. The PA image displays the needle at higher contrast (> 30 dB) than the US image, even at a depth of 12 mm where light energy has largely decayed, demonstrating the potential of the current scanning system for real-time needle guidance. An improved frame rate is envisioned with an optimized scan scheme.

A nanoemulsion contrast agent with a perfluorohexane core and optically absorptive gold nanospheres (GNSs) assembled on the surface, is presented to improve the specificity of photoacoustic (PA) molecular imaging in differentiating targeted cells or aberrant regions from heterogeneous background signals. Compared to distributed GNSs, clustered GNSs at the emulsion oil-water interface produce a red-shifted and broadened absorption spectrum, exhibiting fairly high absorption in the near-infrared region commonly used for deep tissue imaging. Above a certain laser irradiation fluence threshold, a phase transition creating a microbubble in the emulsion core leads to more than 10 times stronger PA signals compared with conventional thermal-expansion-induced PA signals. These signals are also strongly non-linear, as verified by a differential scheme using recorded PA images at different laser fluences. Assuming a linear relation between laser fluence and the PA signal amplitude, differential processing results in nearly perfect suppression of linear sources, but retains a significant residue for the non-linear nanoemulsion with more than 35 dB enhancement. This result demonstrates that contrast specificity can be improved using the nanoemulsion as a targeting agent in PA molecular imaging by suppressing all background signals related to a linear PA response. Furthermore, combined with a system providing simultaneous laser/ultrasound excitation, cavitation-generated bubbles have the potential to be a highly specific contrast agent for ultrasound molecular imaging and harmonic imaging, as well as a targeted means for noninvasive ultrasound-based therapies.

Due to the high scattering coefficient of tissue over the wavelength range used for photoacoustic (PA) imaging, most studies employ bulky, low repetition rate lasers to provide sufficient pulse energies at depth to image within the body. The size and cost of these lasers has impeded integration of photoacoustics into conventional, routinely-used ultrasound (US) scanners. Here, we present an approach leveraging the capabilities of modern, high repetition rate fiber lasers to produce a clinically translatable system providing integrated US/PA images at frame rates > 30 Hz. The system uses a portable, low-cost, low pulse-energy (1 mJ/pulse), high repetition rate (1 kHz), 1064 nm laser and is designed for integrated US/PA imaging of the peripheral vasculature or any relevant diseased region, such as a tumor. Using a rotating galvo-mirror system, the incident laser beam is quickly scanned over the imaging area. Multiple PA images covering the scan area are integrated to form a single PA image. Additionally, ultrasound firings are integrated into the scan sequence to provide an US image reconstructed over the same frame period. We acquired PA images of a 1.5-mm-diameter cylindrical absorber (absorption coefficient 5 cm(-1)) embedded in a tissue-mimicking gelatin phantom at 6-cm depth. A 2 cm x 1 cm (depth x lateral) area was reconstructed. We obtained a signal-to-noise ratio of more than 30 dB, comparable to conventional PA methods using high energy, low repetition rate lasers. The current system produces an integrated US/PA frame at a 32 Hz rate, and 100 Hz frame rates are possible with our present approach.

Optically activated cavitation in a nanoemulsion contrast agent is proposed for therapeutic applications. With a 56°C boiling point perfluorohexane core and highly absorptive gold nanospheres at the oil-water interface, cavitation nuclei in the core can be efficiently induced with a laser fluence below medical safety limits (70 mJ/cm2 at 1064 nm). This agent is also sensitive to ultrasound (US) exposure and can induce inertial cavitation at a pressure within the medical diagnostic range. Images from a high-speed camera demonstrate bubble formation in these nanoemulsions. The potential of using this contrast agent for blood clot disruption is demonstrated in an in vitro study. The possibility of simultaneous laser and US excitation to reduce the cavitation threshold for therapeutic applications is also discussed.

Circulating tumor cells (CTCs) have been reported to correlate most closely with cancer development, and can serve as an important marker for metastatic malignancy, tumor recurrence, and prediction of prognosis and therapeutic effi cacy. Detecting and quantifying CTCs, however, have proven to be challenging due to their low abundance in blood. Based on magneto-optical coupled nanoprobes (made of gold nanorod and iron oxide nanoparticles) and photoacoustic (PA) imaging, we report the development of an enabling technology that can detect CTCs at single cell/mL level. Remarkably, at this low cell concentration, approximately 67% of circulating tumor cells can be captured and imaged with just one pass through the magnetic trapping zone. Compared to the conventional in vitro assays, this technology offers signifi cantly improved sensitivity, because it is inherently compatible with large sample volumes. Compared to more advanced in vivo CTC detection methods, this technology can solve the low throughput problem of optical imaging, and streamline the photoacoustic imaging process by combining magnetic enrichment and digital readout into a single step. Most cancer deaths are caused by metastasis, a process

A new technique using a composite contrast agent is proposed to increase specific contrast in photoacoustic (PA) molecular imaging. The composite particle has an emulsion bead core with a surface-assembled monolayer of gold nanospheres (GNSs), which efficiently absorbs optical energy under laser irradiation and promotes a phase transition of the emulsion bead to form a transient bubble. Much stronger PA signals are generated by the phase transition process than those from thermal expansion, the most common mechanism of laser-sound conversion. By applying a differential scheme of images, recorded at a series of variable fluences, assuming a linear relation between laser fluence and the PA signal amplitude, a nearly perfect suppression of a linear agent can be obtained, while large residues for the nonlinear emulsion beads remain. This result clearly indicates enhanced PA sensitivity and specificity. Localized bubble generation activated by a short laser pulse was also demonstrated using a microscope coupled with a high-speed camera. With simultaneous ultrasound/laser probing, i.e., delivering a probe ultrasound beam after bubble formation, the nonlinear agent shows great potential for ultrasound contrast-enhanced imaging and harmonic imaging.

Photoacoustic (PA) imaging has been demonstrated to be a promising modality in molecular imaging for detection of nanoparticle‐targeted diseased cells or tissues. However, intrinsic absorbers, such as blood, produce strong PA background signals that severely degrade the detection sensitivity and specificity of targeted objects. Magnetomotive photoacoustic (mmPA) imaging, a newly developed molecular imaging modality, introduced dynamic manipulation into traditional PA imaging. Unlike conventional PA imaging, magnetomotive manipulation with simultaneous ultrasound/PA imaging of agents incorporating magnetic nanoparticles enables direct visualization of the signal generating object and can dramatically reduce background signals from strong optical absorbers. This paper briefly reviews recent developments in mmPA imaging, including uses of composite contrast agent, design of magnet system, and data processing for motion filtering. The use of mmPA imaging in detecting rare circulating tumor cells in blood vessels, which remains a big challenge for real‐time in vivo examination using current methodologies, was also addressed. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

The advantages of photoacoustic (PA) imaging, including low cost, non-ionizing operation, and sub-mm spatial resolution at centimeters depth, make it a promising modality to probe nanoparticle-targeted abnormalities in real time at cellular and molecular levels. However, detecting rare cell types in a heterogeneous background with strong optical scattering and absorption remains a big challenge. For example, differentiating circulating tumor cells in vivo (typically fewer than 10 cells/mL for an active tumor) among billions of erythrocytes in the blood is nearly impossible. In this paper, a newly developed technique, magnetomotive photoacoustic (mmPA) imaging, which can greatly increase the sensitivity and specificity of sensing targeted cells or molecular interactions, is reviewed. Its primary advantage is suppression of background signals through magnetic enrichment/manipulation with simultaneous PA detection of magnetic contrast agent targeted objects. Results from phantom and in vitro studies demonstrate the capability of mmPA imaging to differentiate regions targeted with magnetic nanoparticles from the background, and to trap and sensitively detect targeted cells at a concentration of a single cell per milliliter in a flow system mimicking a human peripheral artery. This technique provides an example of the ways in which molecular imaging can potentially enable robust molecular diagnosis and treatment, and accelerate the translation of molecular medicine into the clinic.

Photoacoustic (PA) imaging has been widely used in molecular imaging to detect diseased cells by targeting them with nanoparticle-based contrast agents. However, the sensitivity and specificity are easily degraded because contrast agent signals can be masked by the background. Magnetomotive photoacoustic imaging uses a new type of multifunctional composite particle combining an optically absorptive gold nanorod core and magnetic nanospheres, which can potentially accumulate and concentrate targeted cells while simultaneously enhancing their specific contrast compared to background signals. In this study, HeLa cells molecularly targeted using nanocomposites with folic acid mimicking targeted rare circulating tumor cells (CTCs) were circulated at a 6 ml/min flow rate for trapping and imaging studies. Preliminary results show that the cells accumulate rapidly in the presence of an externally applied magnetic field produced by a dual magnet system. The sensitivity of the current system can reach up to 1 cell/ml in clear water. By manipulating the trapped cells magnetically, the specificity of detecting cells in highly absorptive ink solution can be enhanced with 16.98 dB background suppression by applying motion filtering on PA signals to remove unwanted background signals insensitive to the magnetic field. The results appear promising for future preclinical studies on a small animal model and ultimate clinical detection of rare CTCs in the vasculature.

A new technique using pulsed laser heating of a nanocomposite contrast agent resulting in local bubble formation and concomitant harmonic generation in a scattered probe ultrasound (US) beam is proposed to increase specific contrast in both US imaging and laser-induced photoacoustic (PA) imaging. The composite combines an emulsion bead core with amphiphilic gold nanospheres (GNSs) assembled at the interface. Clustered GNSs result in a broadened absorption spectrum in the near infrared range (700-1000 nm) compared to the typical 520 nm peak of distributed GNSs, enabling their use at depth in tissue. Illuminating the composite with a pulsed laser with appropriately chosen parameters heats the composite through optical absorption by the GNSs and results in a phase transition of the emulsion bead to form a transient bubble. By delivering a probe US pulse simultaneously, or immediately after the laser pulse is delivered, harmonic signals are produced in the scattered US beam. The results show that a residual signal created by subtracting a US signal from the simultaneous US/laser probing signal of the emulsion bead sample is 1.7 dB higher than the laser alone generated PA signal and 20 dB higher than the PA signal of a control homogeneous GNSs dispersion with the same optical absorption, indicating the nonlinear contrast enhancement from bubble dynamics. The proposed technique of local activation of this designed contrast agent can be used to dramatically enhance both the specificity and sensitivity of integrated US/PA molecular imaging.

The possibility of manipulating inertial cavitation induced in a new type of nanoemulsion contrast agent with simultaneous nanosecond pulsed laser and ultrasound illumination is described. The contrast agent consists of an encapsulated emulsion core coated with a layer of 12nm-diameter gold nanospheres. A low-cost, high repetition-rate, low-energy 1064 nm fiber laser transiently heated the emulsion beads to help initiate inertial cavitation due to a phase transition in the emulsion core. It has been shown that inertial cavitation can be initiated at a very low acoustic pressure (0.43MPa) with laser irradiation applied at the rarefaction phase of the incident acoustic wave. The significantly decreased inertial cavitation threshold in this nanoemulsion suggests that it could be an effective tool for site-targeted, molecular therapeutics in addition to its proposed use as a highly specific molecular imaging agent for photoacoustics.

Metastasis, where circulating tumor cells (CTCs) from a primary tumor spread to other organs through the blood or lymphatic system, causes over 90% of cancer deaths.1 Identifying CTCs remains a big challenge due to their rarity in blood. Although many techniques can separate and identify CTCs,2–5 all are applicable in vitro, not in vivo. In addition, they can only sample very limited volumes because repeatedly sampling by blood draws, invasive biopsies, and bone marrow aspirations is unfeasible. In particular, a limited sample volume significantly decreases diagnostic confidence. Furthermore, real-time readout is not possible. Photoacoustic (PA) imaging, which uses ultrasound transducers to detect short laser-pulse-induced acoustic sources, has been used for many biomedical applications.6–8 PA imaging provides high optical contrast in the near IR region and submillimeter acoustic resolution of targets up to several centimeters deep within the body. In recent years, many researchers have shown that by coupling well-designed contrast agents to biological objects, such as tumor cells, targets can be differentiated from the background using PA imaging.9–12 However, when it is necessary to detect targeted cells in blood, the strong PA background signal from blood can easily mask the signals from the contrast agents, especially for rare cell types such as CTCs. We have developed a new imaging technique to suppress these strong background signals through the magnetic accumulation and manipulation of cells (i.e., CTCs) targeted with magneto-sensitive contrast agents. This approach, called magnetomotive photoacoustic (mmPA) imaging, uses nanoparticles with combined strong magnetic and optical Figure 1. Fusion images (ultrasound imaging in grayscale and photoacoustic imaging in color) of beads targeted with composite nanoparticles and trapped in a 12mL/min flow stream at different time points of magnetic action. Triangles indicate the position of the magnet.

Trapping and manipulation of micro-scale objects mimicking metastatic cancer cells in a flow field have been demonstrated with magnetomotive photoacoustic (mmPA) imaging. Coupled contrast agents combining gold nanorods (15 nm × 50 nm; absorption peak around 730 nm) with 15 nm diameter magnetic nanospheres were targeted to 10 μm polystyrene beads recirculating in a 1.6 mm diameter tube mimicking a human peripheral vessel. Targeted objects were then trapped by an external magnetic field produced by a dual magnet system consisting of two disc magnets separated by 6 cm to form a polarizing field (0.04 Tesla in the tube region) to magnetize the magnetic contrast agents, and a custom designed cone magnet array with a high magnetic field gradient (about 0.044 Tesla/mm in the tube region) producing a strong trapping force to magnetized contrast agents. Results show that polystyrene beads linked to nanocomposites can be trapped at flow rates up to 12 ml/min. It is shown that unwanted background in a photoacoustic image can be significantly suppressed by changing the position of the cone magnet array with respect to the tube, thus creating coherent movement of the trapped objects. This study makes mmPA imaging very promising for differential visualization of metastatic cells trafficking in the vasculature.

Magnetic trapping and manipulation of polystyrene beads mimicking magnetically targeted circulating tumor cells (CTCs) has been demonstrated using magnetomotive photoacoustic (mmPA) imaging. These techniques appear promising for the detection of rare CTCs in the vasculature.

The presence of circulating tumor cells (CTCs) in the vasculature directly correlates with the likelihood of metastasis development. Their rarity, however, makes them difficult to detect. Recently, the potential of photoacoustic (PA) imaging for sensitive detection of nanoparticle-targeted diseased cells has been demonstrated. However, the strong blood background can easily mask the weak signal from targeted CTCs. The goal of this study is to utilize a multifunction nanoparticle composite molecularly targeted to CTCs, enabling magnetic trapping for rare cell accumulation with simultaneous PA imaging for detection. This technique greatly increases both the sensitivity and specificity of CTC detection. As an initial test, human prostate cancer cells, LNCaP, targeted with a nanocomposite combining a gold nanorod core with magnetic nanoparticles, circulated in a 1.6-mm tube at a maximum flow rate of 12 ml/min, mimicking CTCs in the human radial artery. Results show that cells can be magnetically trapped, accumulated, and detected at a concentration of only 7 cells/ml, within the typical range of CTCs in peripheral vessels for an active tumor. Future work will focus on improving the sensitivity to detect a single targeted CTC.